HIV-integrase inhibitors, pharmaceutical compositions, and methods for their use

ABSTRACT

Beta-carboline hydroxamic acid compounds represented by formula (I)                  
 
are described. The beta-carboline hydroxamic acid compounds and compositions containing those compounds may be used to inhibit or modulate the activity of HIV integrase enzyme and to treat HIV integrase-mediated diseases and conditions.

The present patent application claims priority to U.S. Ser. No.60/443,223, filed Jan. 27, 2003, which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to beta-carboline hydroxamic acidcompounds and pharmaceutically acceptable salts, pharmaceuticallyacceptable prodrugs, and pharmaceutically acceptable metabolitesthereof, their synthesis, and their use as modulators or inhibitors ofthe human immunodeficiency virus (“HIV”) Integrase enzyme. The compoundsof the present invention are useful for modulating (e.g. inhibiting) anenzyme activity of HIV Integrase enzyme and for treating diseases orconditions mediated by HIV, such as for example, acquiredimmunodeficiency syndrome (“AIDS”), and AIDS related complex (“ARC”).

BACKGROUND OF THE INVENTION

The retrovirus designated “human immunodeficiency virus” or “HIV” is theetiological agent of a complex disease that progressively destroys theimmune system. The disease is known as acquired immune deficiencysyndrome or AIDS. AIDS and other HIV-caused diseases are difficult totreat due to the ability of HIV to rapidly replicate, mutate and acquireresistance to drugs. To attempt to slow the spread of the virus afterinfection, treatment of AIDS and other HIV-caused diseases has focusedon inhibiting HIV replication.

Since HIV is a retrovirus, and thus, encodes a positive-sense RNAstrand, its mechanism of replication is based on the conversion of viralRNA to viral DNA, and subsequent insertion of the viral DNA into thehost cell genome. HIV replication relies on three constitutive HIVencoded enzymes: reverse transcriptase (RT), protease and integrase.

Upon infection with HIV, the retroviral core particles bind to specificcellular receptors and gain entry into the host cell cytoplasm. Onceinside the cytoplasm, viral RT catalyzes the reverse transcription ofviral ssRNA to form viral RNA-DNA hybrids. The RNA strand from thehybrid is then partially degraded and a second DNA strand is synthesizedresulting in viral dsDNA. Integrase, aided by viral and cellularproteins, then transports the viral dsDNA into the host cell nucleus asa component of the pre-integration complex (PIC). In addition, integraseprovides the permanent attachment, i.e., integration, of the viral dsDNAto the host cell genome which, in turn, provides viral access to thehost cellular machinery for gene expression. Following integration,transcription and translation produce viral precursor proteins. Proteasethen cleaves the viral precursor proteins into viral proteins, which,after additional processing, are released from the host cell as newlyinfectious HIV particles.

A key step in HIV replication, insertion of the viral dsDNA into thehost cell genome, is believed to be mediated by integrase in at leastthree, and possibly, four, steps: (1) assembly of proviral DNA; (2)3′-end processing causing assembly of the PIC; (3) 3′-end joining or DNAstrand transfer, i.e., integration; and (4) gap filling, a repairfunction. See, e.g., Goldgur, Y. et al., PNAS 96(23): 13040–13043(November 1999); Sayasith, K. et al., Expert Opin. Ther. Targets 5(4):443–464 (2001); Young, S. D., Curr. Opin. Drug Disc. & Devel. 4(4):402–410 (2001); Wai, J. S. et al, J. Med. Chem. 43(26): 4923–4926(2000); Debyser, Z. et al., Assays for the Evaluation of HIV-1 IntegraseInhibitors, from Methods in Molecular Biology 160: 139–155, Schein, C.H. (ed.), Humana Press Inc., Totowa, N.J. (2001); and Hazuda, D. et al.,Drug Design and Disc. 13: 17–24 (1997).

In the first step, integrase forms a stable complex with the viral longterminal repeat (LTR) regions. Once the complex is formed, integrasethen performs an endonucleolytic processing step whereby the terminal GTdinucleotides of the 3′ ends (immediately downstream from a conserved CAdinucleotide) of both DNA strands are cleaved. The processedDNA/integrase complex (the PIC) then translocates across the nuclearmembrane. Once inside the host cell nucleus, integrase performs thethird step, 3′-end joining, whereby a cut is made in the host cell DNAto covalently join the processed 3′-ends of the viral processed DNAduring two transesterification reactions. In the fourth step, cellularenzymes repair the resultant gap at the site of viral DNA insertion. Theenzymes, if any, employed in the repair process have not been accuratelyidentified. Sayasith, K. et al., Expert Opin. Ther. Targets 5(4):443–464 (2001). Thus, the role that integrase plays in the gap fillingfunction is not known.

It is clear that the role that integrase plays in the integration of theviral DNA into the host cell genome occurs through well-orderedreactions directed by various viral and cellular factors. This knowledgeprovides a variety of opportunities to block the essential step ofintegration (and the essential enzyme integrase) in the HIV life cycle.

Currently, AIDS and other HIV-caused disease are treated with an “HIVcocktail” containing multiple drugs including RT and proteaseinhibitors. However, numerous side effects and the rapid emergence ofdrug resistance limit the ability of the RT and protease inhibitors tosafely and effectively treat AIDS and other HIV-caused diseases. In viewof the shortcomings of RT and protease inhibitors, there is a need foranother mechanism through which HIV replication can be inhibited.Integration, and thus integrase, a virally encoded enzyme with nomammalian counterpart, is a logical alternative. See, e.g., Wai, J. S.et al., J. Med. Chem. 43: 4923–4926 (2000); Grobler, J. et al., PNAS 99:6661–6666 (2002); Pais, G. C. G. et al, J. Med. Chem. 45:3184–3194(2002); Young, S. D., Curr. Opin. Drug Disc. & Devel. 4(4):402–410 (2001); Godwin, C. G. et al, J. Med. Chem. 45: 3184–3194 (2002);Young, S. D. et al., “L-870,810: Discovery of a Potent HIV IntegraseInhibitor with Potential Clinical Utility,” Poster presented at the XIVInternational AIDS Conference, Barcelona (Jul. 7–12, 2002); and WO02/070491.

It has been suggested that for an integrase inhibitor to function, itshould inhibit the strand transfer integrase function. See, e.g., Young,S. D., Curr. Opin. Drug Disc. & Devel. 4(4): 402–410 (2001). Thus, thereis a need for HIV inhibitors, specifically, integrase inhibitors, and,more specifically, strand transfer inhibitors, to treat AIDS and otherHIV-caused diseases. The inventive agents disclosed herein are novel,potent and selective HIV-integrase inhibitors, and, more specifically,strand transfer inhibitors, with high antiviral activity and lowtoxicity.

The references made to published documents throughout this applicationmore fully describe the state of the art to which this inventionpertains. The disclosures of these references are hereby incorporated byreference in their entireties.

SUMMARY OF THE INVENTION

The invention is directed to compounds represented by Formula I:

wherein:

R₁, R₂, R₃, R₄, R₅, R₆ independently are selected from the groupconsisting of: hydrogen; halogen; and a lower alkyl, lower alkoxy alkyl,lower alkenyl, lower alkynyl, OR_(c), and N(R_(c))₂ group, unsubstitutedor substituted with one or more halogens, where R_(c) is hydrogen;oxygen; or an unsubstituted lower alkyl, unsubstituted lower alkenyl, orunsubstituted lower alkynyl group;

R₇ is a lower alkyl, lower alkenyl, lower alkynyl, or —O—, unsubstitutedor substituted with one or more substituents independently selected fromthe group consisting of:

-   -   hydrogen; halogens; a lower alkyl, lower alkenyl, lower alkynyl,        aryl, cycloalkyl, heterocycloalkyl, and heteroaryl group; and        —O—, unsubstituted or substituted with one or more substituents        independently selected from the group consisting of:        -   halogens; hydrogen; and a lower alkyl, lower alkenyl, lower            alkynyl, aryl, cycloalkyl, heterocycloalkyl, and heteroaryl            group, unsubstituted or substituted with one or more            halogens;

R₈ and R₉ independently are selected from the group consisting of:hydrogen; and an alkyl, alkenyl, and alkynyl group, unsubstituted orsubstituted with one or more substituents independently selected fromthe group consisting of:

-   -   halogens; and an aryl, cycloalkyl, heterocycloalkyl, and        heteroaryl group, unsubstituted or substituted with one or more        substituents independently selected from the group consisting        of:        -   halogens; and an unsubstituted lower alkyl, unsubstituted            lower alkenyl, and unsubstituted lower alkynyl group; and

R₂ and R₈ together with the N to which R₈ is attached cyclize to formthe following compound represented by the Formula Ib:

wherein R₁₀ and R₁₁ are each independently:

-   -   hydrogen; halogen; a lower alkyl, lower alkenyl, lower alkynyl,        OR_(c), or N(R_(c))₂ group, unsubstituted or substituted with        one or more substituents independently selected from the group        consisting of:        -   halogens; and an aryl, cycloalkyl, heterocycloalkyl, and            heteroaryl group, unsubstituted or substituted with one or            more substituents independently selected from the group            consisting of:            -   halogens; and an unsubstituted lower alkyl,                unsubstituted lower alkenyl, and unsubstituted lower                alkynyl group;    -   where R_(c) is halogen; hydrogen; oxygen; or an unsubstituted        lower alkyl, unsubstituted lower alkenyl, or unsubstituted lower        alkynyl group; and    -   n is 1, 2 or 3.

The present invention also provides compounds of Formula (I),

wherein:

R₁, R₂, R₃, R₄, R₅, and R₆ are independently selected from hydrogen,halogen, C₁₋C₆ alkyl, alkoxy C₁–C₆ alkyl, C₂–C₆ alkenyl, C₂–C₆ alkynyl,—OR_(c), —NO₂, and —N(R_(c))₂;

each R_(c) is independently selected from hydrogen, C₁–C₆ alkyl, C₂–C₆alkenyl, and C₂–C₆ alkynyl;

R₇ is C₁–C₆ alkyl, C₂–C₆ alkenyl, or C₂–C₆ alkynyl, all of which areoptionally substituted by one or more substituents independentlyselected from halogen, C₁–C₆ alkyl, C₂–C₆ alkenyl, C₂–C₆ alkynyl, aryl,cycloalkyl, heterocycloalkyl, and heteroaryl, wherein said aryl,cycloalkyl, and heterocycloalkyl are optionally substituted with one ormore substituents independently selected from halogen, C₁–C₆ alkyl,C₂–C₆ alkenyl, and C₂–C₆ alkynyl;

R₈ and R₉ are independently selected from hydrogen, C₁–C₆ alkyl, C₂–C₆alkenyl, and C₂–C₆ alkynyl, wherein said alkyl, alkenyl, and alkynyl areoptionally substituted with one or more substituents independentlyselected from halogen, aryl, cycloalkyl, heterocycloalkyl, andheteroaryl group, wherein said aryl, cycloalkyl, and heterocycloalkylare optionally substituted with one or more substituents independentlyselected from halogen, C₁–C₆ alkyl, C₂–C₆ alkenyl, and C₂–C₆ alkynyl;and

pharmaceutically acceptable salts and solvates thereof.

In another aspect of the present invention are provided compounds ofFormula (I), wherein:

R₁, R₂, R₃, R₄, R₅, and R₆ are independently selected from hydrogen,—N(R_(c))₂, and —NO₂; and

R_(c) is selected from hydrogen, C₁–C₆ alkyl, C₂–C₆ alkenyl, and C₂–C₆alkynyl; and

pharmaceutically acceptable salts and solvates thereof.

In yet another aspect of the present invention are provided compounds offormula (I) wherein R₇ is C₁–C₆ alkyl, optionally substituted with aryl,cycloalkyl, heterocycloalkyl, and heteroaryl, wherein said aryl,cycloalkyl, heterocycloalkyl, and heteroaryl are optionally substitutedwith at least one substituent selected from halogen, C₁–C₆ alkyl, C₂–C₆alkenyl, and C₂–C₆ alkynyl; and pharmaceutically acceptable salts andsolvates thereof.

In still another aspect of the present invention are provided compoundsof formula (I), wherein R₈ and R₉ are independently selected fromhydrogen and C₁–C₆ alkyl, wherein said alkyl group is optionallysubstituted with aryl, and wherein said aryl is optionally substitutedwith at least one substituent selected from halogen and C₁–C₆ alkyl; andpharmaceutically acceptable salts and solvates thereof.

In still a further aspect of the present invention are provided compoundof formula (I), wherein:

R₁, R₂, R₃, R₄, R₅, and R₆ are independently selected from hydrogen and—N(R_(c))₂;

R_(c) is hydrogen, C₁–C₆ alkyl, C₂–C₆ alkenyl, and C₂–C₆ alkynyl;

R₇ is C₁–C₆ alkyl, optionally substituted with at least one substituentselected from aryl, cycloalkyl, heterocycloalkyl, and heteroaryl,wherein said aryl, cycloalkyl, heterocycloalkyl, and heteroaryl areoptionally substituted with at least one substituent selected fromhalogen, C₁–C₆ alkyl, C₂–C₆ alkenyl, and C₂–C₆ alkynyl;

R₈ and R₉ are independently selected from hydrogen and C₁–C₆ alkyl,wherein said alkyl is optionally substituted with at least onesubstituent selected from aryl group, wherein said aryl is optionallysubstituted with at least one substituent selected from halogen, andC₁–C₆ alkyl; and

pharmaceutically acceptable salts and solvates thereof.

In yet another aspect of the invention are provided compounds of formula(I), wherein:

R₁, R₂, R₃, R₄, R₅, R₆ are independently selected from hydrogen, —NH₂;and —NO₂;

R₇ is 4-fluorobenzyl, (5-chlorothien-2-yl)methyl,3-chloro-2-fluorobenzyl, benzyl, 4-methylbenzyl, 2,4-difluorobenzyl,3-chloro-2,6-difluorobenzyl, or 3-chlorobenzyl; and

R₈ and R₉ are independently selected from hydrogen, methyl, and benzyl;and

pharmaceutically acceptable salts and solvates thereof.

Another aspect of the present invention provides compounds of formula(I), wherein:

R₁, R₂, R₃, R₄, R₅ and R₆ are hydrogen;

R₇ is —CH₂phenyl, wherein said phenyl is substituted with at least onesubstitutent chosen from fluorine and chlorine;

R₈ is hydrogen or —CH₃;

R₉ is hydrogen or —CH₃; and

pharmaceutically acceptable salts and solvates thereof.

In still another aspect of the present invention are provided compoundsof formula (I), wherein:

R₁, R₂, R₃, R₅ and R₆ are hydrogen;

R₄ is —NO₂ or —NH₂;

R₇ is —CH₂phenyl, wherein said phenyl is substituted with at least onesubtitutent chosen from fluorine and chlorine;

R₈ is hydrogen or —CH₃;

R₉ is hydrogen or —CH₃; and

pharmaceutically acceptable salts and solvates thereof.

In yet another aspect of the present invention are provided compounds offormula (I), wherein:

R₁, R₂, R₃, R₄, R₅ and R₆ are hydrogen;

R₇ is —CH₂phenyl, wherein said phenyl is substituted with at least onesubtitutent chosen from fluorine and chlorine;

R₈ and R₉ are hydrogen; and

pharmaceutically acceptable salts and solvates thereof.

Another aspect of the present invention provides compounds of formula(I), wherein:

R₁, R₂, R₃, R₄, R₅ and R₆ are hydrogen;

R₇ is —CH₂phenyl, wherein said phenyl is substituted with at least onesubtitutent chosen from fluorine and chlorine;

R₈ and R₉ are —CH₃; and

pharmaceutically acceptable salts and solvates thereof.

Another aspect of the present invention provides compounds of formula(I), wherein:

R₁, R₂, R₃, R₄, R₅ and R₆ are hydrogen;

R₇ is —CH₂phenyl, wherein said phenyl is substituted with at least onesubtitutent chosen from fluorine and chlorine;

R₈ is hydrogen;

R₉ is —CH₃; and

pharmaceutically acceptable salts and solvates thereof.

Another aspect of the present invention provides compounds of formula(I), wherein:

R₁, R₂, R₃, R₄, R₅ and R₆ are hydrogen;

R₇ is —CH₂phenyl, wherein said phenyl is substituted with at least onesubtitutent chosen from fluorine and chlorine;

R₈ is —CH₃;

R₉ is hydrogen; and

pharmaceutically acceptable salts and solvates thereof.

In another aspect of the present invention, the compounds of formula (I)are selected from9-(4-Fluorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide;9-[(5-Chlorothien-2-yl)methyl]-N-hydroxy-9H-β-carboline-3-carboxamide;9-(3-Chloro-2-fluorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide;9-Benzyl-N-hydroxy-9H-β-carboline-3-carboxamide;9-(4-Methylbenzyl)-N-Hydroxy-9H-β-carboline-3-carboxamide;9-(2,4-Difluorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide;9-(3-Chloro-2,6-difluorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide;6-Amino-9-(3-chlorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide;9-(3-Chloro-2,6-difluorobenzyl)-N-methoxy-9H-β-carboline-3-carboxamide;N-(Benzyloxy)-9-(3-chloro-2,6-difluorobenzyl)-9H-β-carboline-3-carboxamide;9-(3-Chloro-2,6-difluorobenzyl)-N-hydroxy-N-methyl-9H-β-carboline-3-carboxamide;N-Benzyl-9-(3-chloro-2,6-difluorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide;9-(4-fluorobenzyl)-N-hydroxy-N-methyl-9H-β-carboline-3-carboxamide; andpharmaceutically acceptable salts and solvates thereof.

In another aspect of the present invention are provided compounds offormula (Ib),

wherein:

R₁, R₃, R₄, R₅, and R₆ are independently selected from hydrogen,halogen, C₁–C₆ alkyl, alkoxy C₁–C₆ alkyl, C₂–C₆ alkenyl, C₂–C₆ alkynyl,—OR_(c), —NO₂, and —N(R_(c))₂;

each R_(c) is independently selected from hydrogen, C₁–C₆ alkyl, C₂–C₆alkenyl, and C₂–C₆ alkynyl;

R₇ is C₁–C₆ alkyl, C₂–C₆ alkenyl, or C₂–C₆ alkynyl, all of which areoptionally substituted by one or more substituents independentlyselected from halogen, C₁–C₆ alkyl, C₂–C₆ alkenyl, C₂–C₆ alkynyl, aryl,cycloalkyl, heterocycloalkyl, and heteroaryl, wherein said aryl,cycloalkyl, and heterocycloalkyl are optionally substituted with one ormore substituents independently selected from halogen, C₁–C₆ alkyl,C₂–C₆ alkenyl, and C₂–C₆ alkynyl;

R₉ is independently selected from hydrogen, C₁–C₆ alkyl, C₂–C₆ alkenyl,and C₂–C₆ alkynyl, wherein said alkyl, alkenyl, and alkynyl areoptionally substituted with one or more substituents independentlyselected from halogen, aryl, cycloalkyl, heterocycloalkyl, andheteroaryl group, wherein said aryl, cycloalkyl, and heterocycloalkylare optionally substituted with one or more substituents independentlyselected from halogen, C₁–C₆ alkyl, C₂–C₆ alkenyl, and C₂–C₆ alkynyl;

each R₁₀ and R₁₁ are independently selected from hydrogen, halogen,C₁–C₆ alkyl, C₂–C₆ alkenyl, and C₂–C₆ alkynyl, —OR_(c), or —N(R_(c))₂group, wherein said alkyl, alkenyl, and alkynyl are optionallysubstituted by one or more substituents selected from halogen, aryl,cycloalkyl, heterocycloalkyl, and heteroaryl group, wherein said aryl,cycloalkyl, heterocycloalkyl, and heteroaryl are optionally substitutedwith at least one substitutent independently selected from halogen,C₁–C₆ alkyl, C₂–C₆ alkenyl, and C₂–C₆ alkynyl;

n is 1, 2 or 3; and

pharmaceutically acceptable salts and solvates thereof.

In addition to compounds of formula (I) and (Ib), the invention is alsodirected to pharmaceutically acceptable salts, pharmaceuticallyacceptable prodrugs, and pharmaceutically active metabolites of suchcompounds, and pharmaceutically acceptable salts of such metabolites.Such compounds, salts, prodrugs and metabolites are at timescollectively referred to herein as “HIV Integrase agents.”

The invention also relates to pharmaceutical compositions, eachcomprising a therapeutically effective amount of at least one HIVIntegrase agent according to the invention and a pharmaceuticallyacceptable carrier, diluent, or vehicle therefore.

Additionally, the invention is directed to methods of inhibiting ormodulating an enzyme activity of human immunodeficiency virus (HIV)integrase, comprising contacting said enzyme with an effective amount ofat least one HIV Integrase agent of the invention.

In another aspect, the invention is directed to methods of treating adisease or condition mediated by human immunodeficiency virus (HIV)integrase enzyme, comprising administering to a mammal in need of suchtreatment a therapeutically effective amount of at least one HIVIntegrase agent of the invention. The disease or condition may be, forexample, acquired immunodeficiency syndrome (AIDS) or AIDS relatedcomplex (ARC).

In a further aspect of the present invention are provided methods forinhibiting the replication of human immunodeficiency virus (HIV) in amammal, comprising administering a human immunodeficiency virusinhibiting amount of a compound of formula (I) or (Ib), or apharmaceutically acceptable salt or solvate thereof, to said mammal.

In another aspect of the present invention are provided methods ofinhibiting the activity of the HIV integrase enzyme, comprisingcontacting said enzyme with a HIV integrase enzyme-inhibiting amount ofa compound of formula (I) or (Ib), or a pharmaceutically acceptable saltor solvate thereof. Also provided in the present invention are thosemethods of inhibiting the activity of the HIV integrase enzyme, whereinthe enzyme is found in a mammal.

A further aspect of the present invention provides a medicament,comprising a compound of formula (I) or (Ib), or a pharmaceuticallyacceptable salt or solvate thereof, for the treatment of a disease orcondition mediated by human immunodeficiency virus (HIV) integraseenzyme.

As used herein, the terms “comprising” and “including” are used hereinin their open, non-limiting sense.

The term “alkyl” refers to a straight- or branched-chain alkyl grouphaving from 1 to 12 carbon atoms in the chain. Exemplary alkyl groupsinclude methyl (Me, which also may be structurally depicted by “/”),ethyl (Et), n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl(tBu), pentyl, isopentyl, neo-pentyl, hexyl, isohexyl, and the like.

The term “heteroalkyl” refers to a straight- or branched-chain alkylgroup having from 2 to 12 atoms in the chain, one or more of which is aheteroatom selected from S, O, and N. Exemplary heteroalkyls includealkyl ethers, secondary and tertiary alkyl amines, alkyl sulfides, andthe like.

The term “alkenyl” refers to a straight- or branched-chain alkenyl grouphaving from 2 to 12 carbon atoms in the chain. Illustrative alkenylgroups include prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl,hex-2-enyl, and the like.

The term “alkynyl” refers to a straight- or branched-chain alkynyl grouphaving from 2 to 12 carbon atoms in the chain. Illustrative alkynylgroups include prop-2-ynyl, but-2-ynyl, but-3-ynyl, 2-methylbut-2-ynyl,hex-2-ynyl, and the like.

The terms “lower alkyl”, “lower alkenyl”, and “lower alkynyl” refer,respectively, to an alkyl, alkenyl, and alkynyl group having from one(1) to six (6) carbon atoms in the chain.

The term “haloalkyl” refers to a straight- or branched-chain alkyl,alkenyl or alkynyl group having from 2–12 carbon atoms in the chain andwhere one or more hydrogens is replaced with a halogen. Illustrativehaloalkyl groups include trifluoromethyl, 2-bromopropyl, 3-chlorohexyl,1-iodo-isobutyl, and the like.

The term “aryl” (Ar) refers to a monocyclic, or fused or spiropolycyclic, aromatic carbocycle (ring structure having ring atoms thatare all carbon) having from 3 to 12 ring atoms per ring. Illustrativeexamples of aryl groups include the following moieties:

and the like.

The term “heteroaryl” (heteroAr) refers to a monocyclic, or fused orspiro polycyclic, aromatic heterocycle (ring structure having ring atomsselected from carbon atoms as well as nitrogen, oxygen, and sulfurheteroatoms) having from 3 to 12 ring atoms per ring. Illustrativeexamples of aryl groups include the following moieties:

and the like.

The term “cycloalkyl” refers to a saturated or partially saturated,monocyclic or fused or spiro polycyclic, carbocycle having from 3 to 12ring atoms per ring. Illustrative examples of cycloalkyl groups includethe following moieties:

and the like.

The term “heterocycloalkyl” refers to a monocyclic, or fused or spiropolycyclic, ring structure that is saturated or partially saturated andhas from 3 to 12 ring atoms per ring selected from C atoms and N, O, andS heteroatoms. Illustrative examples of heterocycloalkyl groups include:

and the like.

The term “halogen(s)” represents chlorine, fluorine, bromine or iodine.The term “halo” represents chloro, fluoro, bromo or iodo.

The term “substituted” means that the specified group or moiety bearsone or more substituents. The term “unsubstituted” means that thespecified group bears no substituents.

The term “optionally substituted” means that the specified group isunsubstituted or substituted by one or more substituents.

The term “pharmaceutically acceptable salts” refers to salt forms thatare pharmacologically acceptable and substantially non-toxic to thesubject being administered the HIV Integrase agent.

A “therapeutically effective amount” is intended to mean that amount ofa compound that, when administered to a mammal in need of suchtreatment, is sufficient to effect treatment, as defined herein. Thus,e.g., a therapeutically effective amount of a compound of the Formula I,salt, active metabolite or prodrug thereof, is a quantity sufficient tomodulate or inhibit the activity of HIV Integrase such that a diseasecondition that is mediated by activity is reduced or alleviated.

The terms “treat”, “treating”, and “treatment” refer to any treatment ofa HIV Integrase mediated disease or condition in a mammal, particularlya human, and include: (i) preventing the disease or condition fromoccurring in a subject which may be predisposed to the condition, suchthat the treatment constitutes prophylactic treatment for the pathologiccondition; (ii) modulating or inhibiting the disease or condition, i.e.,arresting its development; (iii) relieving the disease or condition,i.e., causing regression of the disease or condition; or (iv) relievingand/or alleviating the disease or condition or the symptoms resultingfrom the disease or condition, e.g., relieving an inflammatory responsewithout addressing the underlying disease or condition.

The term “human immunodeficiency virus-inhibiting amount,” as usedherein, refers to the amount of a compound of the present invention, ora pharmaceutically acceptable salt of solvate thereof, required toinhibit replication of the human immunodeficiency virus (HIV) in vivo,such as in a mammal, or in vitro. The amount of such compounds requiredto cause such inhibition can be determined without undue experimentationusing methods known to those of ordinary skill in the art and thosedescribed herein.

The term, “HIV integrase enzyme-inhibiting amount,” as used hereinrefers to the amount of a compound of the present invention, or apharmaceutically acceptable salt or solvate thereof, required todecrease the activity of the HIV integrase enzyme either in vivo, suchas in a mammal, or in vitro. Such inhibition can take place bycontacting the HIV integrase enzyme with a compound of the presentinvention, or a pharmaceutically acceptable salt or solvate thereof.Such inhibition may take place by the compound of the present inventionbinding directly to the HIV integrase enzyme. In addition, the activityof the HIV integrase enzyme may be decreased in the presence of acompound of the present invention when such direct binding between theenzyme and the compound does not take place. Furthermore, suchinhibition may be competitive, non-competitive, or uncompetitive. Suchinhibition may be determined using in vitro or in vivo systems, or acombination of both, using methods known to those of ordinary skill inthe art.

The term, “compound of the present invention” refers to compounds offormula (I) or (Ib), or pharmaceutically acceptable salts or solvatesthereof.

DETAILED DESCRIPTION

The compounds of the present invention are useful for modulating orinhibiting HIV Integrase enzyme. More particularly, the compounds of thepresent invention are useful as modulators or inhibitors of HIVIntegrase activity, and thus are useful for the prevention and/ortreatment of HIV mediated diseases or conditions (e.g., AIDS, and ARC),alone or in combination with other known antiviral agents.

The compounds of the present invention may have asymmetric carbon atoms.The carbon-carbon bonds in the compounds of the present invention may bedepicted herein using a solid line (

), a solid wedge (

), or a dotted wedge (

). The use of a solid line to depict bonds to asymmetric carbon atoms ismeant to indicate that all possible stereoisomers at that carbon atomare included. The use of either a solid or dotted wedge to depict bondsto asymmetric carbon atoms is meant to indicate that only thestereoisomer shown is meant to be included. It is possible thatcompounds of the invention may contain more than one asymmetric carbonatom. In those compounds, the use of a solid line to depict bonds toasymmetric carbon atoms is meant to indicate that all possiblestereoisomers are meant to be included. The use of a solid line todepict bonds to one or more asymmetric carbon atoms in a compound of theinvention and the use of a solid or dotted wedge to depict bonds toother asymmetric carbon atoms in the same compound is meant to indicatethat a mixture of diastereomers is present.

Individual enantiomers of the compounds of the present invention can bedesignated as either the (R)- or (S)-enantiomer using conventionalnaming protocols known to those of ordinary skill in the art and asdescribed in E. L. Eliel et al., Stereochemistry of Organic Compounds,Wiley: New York, 1994. Furthermore, when a compound of the presentinvention contains more than one chiral carbon atom, the stereochemistryof the individual carbon atoms may be assigned as of either the (R)- or(S)-configuration according to methods known to those of ordinary skillin the art and as described in E. L. Eliel et al., Stereochemistry ofOrganic Compounds, Wiley: New York, 1994.

Solutions of individual stereoisomeric compounds of the presentinvention may rotate plane-polarized light. The use of either a “(+)” or“(−)” symbol in the name of a compound of the invention indicates that asolution of a particular stereoisomer rotates plane-polarized light inthe (+) or (−) direction, as measured using techniques known to those ofordinary skill in the art and as described in E. L. Eliel et al.,Stereochemistry of Organic Compounds, Wiley: New York, 1994.

Diastereomeric mixtures can be separated into their individualdiastereomers on the basis of their physical chemical differences bymethods known to those skilled in the art, for example, bychromatography or fractional crystallization. Enantiomers can beseparated by converting the enantiomeric mixtures into a diastereomericmixture by reaction with an appropriate optically active compound (e.g.,alcohol), separating the diastereomers and converting (e.g.,hydrolyzing) the individual diastereomers to the corresponding pureenantiomers. Other methods of separating individual diastereomericcompounds are described in E. L. Eliel et al., Stereochemistry ofOrganic Compounds, Wiley: New York, 1994. All such isomers, includingenantiomeric mixtures, diastereomeric mixtures, and pure enantiomers areconsidered part of the present invention.

Alternatively, individual stereoisomeric compounds of the presentinvention may be prepared in enantiomerically enriched form byasymmetric synthesis, followed by purification as described above ifnecessary. Asymmetric synthesis may be performed using techniques knownto those of ordinary skill in the art, such as the use of asymmetricstarting materials that are commercially available or readily preparedusing methods known to those of ordinary skill in the art, the use ofasymmetric auxiliaries that may be removed at the completion of thesynthesis, or the resolution of intermediate compounds using enzymaticmethods. Other methods of preparing enantiomerically pure compounds aredescribed in E. L. Eliel et al., Stereochemistry of Organic Compounds,Wiley: New York, 1994. The choice of which method is used will depend onfactors that include, but are not limited to, the availability ofstarting materials, the relative efficiency of a method, and whethersuch methods are useful for the compounds of the invention containingparticular functional groups. Such choices are within the knowledge ofone of ordinary skill in the art.

When the compounds of the present invention contain asymmetric carbonatoms, the compounds, pharmaceutically acceptable salts or solvates mayexist as single stereoisomers, racemates, and/or mixtures of enantiomersand/or diastereomers. All such single stereoisomers, racemates, andmixtures thereof are intended to be within the scope of the presentinvention.

It is understood that while a compound may exhibit the phenomenon oftautomerism, the formula drawings within this specification expresslydepict only one of the possible tautomeric forms. It is therefore to beunderstood that a formula is intended to represent any tautomeric formof the depicted compound and is not to be limited merely to a specificcompound form depicted by the structural formula.

It is also understood that a compound of the present invention may existas an “E” or “Z” configurational isomer, or a mixture of E and Zisomers. It is therefore to be understood that a formula is intended torepresent any configurational form of the depicted compound and is notto be limited merely to a specific compound form depicted by the formuladrawings.

Some of the inventive compounds may exist as single stereoisomers (i.e.,essentially free of other stereoisomers), racemates, and/or mixtures ofenantiomers and/or diastereomers. All such single stereoisomers,racemates and mixtures thereof are intended to be within the scope ofthe present invention. In one embodiment, the inventive compounds thatare optically active are used in optically pure form.

As generally understood by those skilled in the art, an optically purecompound having one chiral center (i.e., one asymmetric carbon atom) isone that consists essentially of one of the two possible enantiomers(i.e., is enantiomerically pure), and an optically pure compound havingmore than one chiral center is one that is both diastereomerically pureand enantiomerically pure. Preferably, the compounds of the presentinvention are used in a form that is at least 90% optically pure, thatis, a form that contains at least 90% of a single isomer (80%enantiomeric excess (“e.e.”) or diastereomeric excess (“d.e.”)), morepreferably at least 95% (90% e.e. or d.e.), even more preferably atleast 97.5% (95% e.e. or d.e.), and most preferably at least 99% (98%e.e. or d.e.).

Additionally, formulae (I) and (Ib) are intended to cover, whereapplicable, solvated as well as unsolvated forms of the compounds. Thus,each formula includes compounds having the indicated structure,including the hydrated as well as the non-hydrated forms.

In addition to compounds of the present invention, the HIV Integraseagents of the invention include pharmaceutically acceptable salts,prodrugs, and active metabolites of such compounds, and pharmaceuticallyacceptable salts of such metabolites. A “pharmaceutically acceptableprodrug” is a compound that may be converted under physiologicalconditions or by solvolysis to the specified compound or to apharmaceutically acceptable salt of such compound. A “pharmaceuticallyactive metabolite” is a pharmacologically active product producedthrough metabolism in the body of a specified compound or salt thereof.Prodrugs and active metabolites of a compound may be identified usingroutine techniques known in the art. See, e.g., Bertolini et al., J.Med. Chem., 40, 2011–2016 (1997); Shan et al., J. Pharm. Sci., 86(7),765–767 (1997); Bagshawe, Drug Dev. Res., 34, 220–230 (1995); Bodor,Advances in Drug Res., 13, 224–331 (1984); Bundgaard, Design of Prodrugs(Elsevier Press 1985); Larsen, Design and Application of Prodrugs, DrugDesign and Development (Krogsgaard-Larsen et al. eds., Harwood AcademicPublishers, 1991); Dear et al., J. Chromatogr. B, 748, 281–293 (2000);Spraul et al., J. Pharmaceutical & Biomedical Analysis; 10(8), 601–605(1992); and Prox et al., Xenobiol., 3(2), 103–112 (1992).

If a derivative used in the method of the invention is a base, a desiredsalt may be prepared by any suitable method known to the art, includingtreatment of the free base with an inorganic acid, such as hydrochloricacid; hydrobromic acid; sulfuric acid; nitric acid; phosphoric acid; andthe like, or with an organic acid, such as acetic acid; maleic acid;succinic acid; mandelic acid; fumaric acid; malonic acid; pyruvic acid;oxalic acid; glycolic acid; salicylic acid; pyranosidyl acid, such asglucuronic acid or galacturonic acid; alpha-hydroxy acid, such as citricacid or tartaric acid; amino acid, such as aspartic acid or glutamicacid; aromatic acid, such as benzoic acid or cinnamic acid; sulfonicacid, such as p-toluenesulfonic acid or ethanesulfonic acid; and thelike.

If a derivative used in the method of the invention is an acid, adesired salt may be prepared by any suitable method known to the art,including treatment of the free acid with an inorganic or organic base,such as an amine (primary, secondary, or tertiary); an alkali metal oralkaline earth metal hydroxide; or the like. Illustrative Examples ofsuitable salts include organic salts derived from amino acids such asglycine and arginine; ammonia; primary, secondary, and tertiary amines;and cyclic amines, such as piperidine, morpholine, and piperazine; aswell as inorganic salts derived from sodium, calcium, potassium,magnesium, manganese, iron, copper, zinc, aluminum, and lithium.

A “solvate” is intended to mean a pharmaceutically acceptable solvateform of a specified compound that retains the biological effectivenessof such compound. Examples of solvates include, but are not limited to,compounds of the invention in combination with water, isopropanol,ethanol, methanol, dimethylsulfoxide (DMSO), ethyl acetate, acetic acid,ethanolamine, or mixtures thereof.

A “pharmaceutically acceptable salt” is intended to mean a salt thatretains the biological effectiveness of the free acids and bases of thespecified derivative, containing pharmacologically acceptable anions,and is not biologically or otherwise undesirable. Examples ofpharmaceutically acceptable salts include, but are not limited to,acetate, acrylate, benzenesulfonate, benzoate (such as chlorobenzoate,methylbenzoate, dinitrobenzoate, hydroxybenzoate, and methoxybenzoate),bicarbonate, bisulfate, bisulfite, bitartrate, borate, bromide,butyne-1,4-dioate, calcium edetate, camsylate, carbonate, chloride,caproate, caprylate, clavulanate, citrate, decanoate, dihydrochloride,dihydrogenphosphate, edetate, edislyate, estolate, esylate,ethylsuccinate, formate, fumarate, gluceptate, gluconate, glutamate,glycollate, glycollylarsanilate, heptanoate, hexyne-1,6-dioate,hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,γ-hydroxybutyrate, iodide, isobutyrate, isothionate, lactate,lactobionate, laurate, malate, maleate, malonate, mandelate, mesylate,metaphosphate, methane-sulfonate, methylsulfate, monohydrogenphosphate,mucate, napsylate, naphthalene-1-sulfonate, naphthalene-2-sulfonate,nitrate, oleate, oxalate, pamoate (embonate), palmitate, pantothenate,phenylacetates, phenylbutyrate, phenylpropionate, phthalate,phospate/diphosphate, polygalacturonate, propanesulfonate, propionate,propiolate, pyrophosphate, pyrosulfate, salicylate, stearate,subacetate, suberate, succinate, sulfate, sulfonate, sulfite, tannate,tartrate, teoclate, tosylate, triethiodode, and valerate salts.

It is understood by those of ordinary skill in the art that thecompounds of the present invention, or their pharmaceutically acceptablesalts or solvates, may exist in different polymorph or crystal forms,all of which are intended to be within the scope of the presentinvention and specified formulas. In addition, the compounds of thepresent invention, and their pharmaceutically acceptable salts andsolvates, may exist as tautomers, all of which are intended to be withinthe broad scope of the present invention.

The compounds of the present invention that are basic in nature arecapable of forming a wide variety of different salts with variousinorganic and organic acids. Although such salts must bepharmaceutically acceptable for administration to animals, it is oftendesirable in practice to initially isolate the compound of the presentinvention from the reaction mixture as a pharmaceutically unacceptablesalt and then simply convert the latter back to the free base compoundby treatment with an alkaline reagent and subsequently convert thelatter free base to a pharmaceutically acceptable acid addition salt.The acid addition salts of the base compounds of this invention can beprepared by treating the base compound with a substantially equivalentamount of the selected mineral or organic acid in an aqueous solventmedium or in a suitable organic solvent, such as methanol or ethanol.Upon evaporation of the solvent, the desired solid salt is obtained. Thedesired acid salt can also be precipitated from a solution of the freebase in an organic solvent by adding an appropriate mineral or organicacid to the solution.

Those compounds of the present invention that are acidic in nature arecapable of forming base salts with various pharmacologically acceptablecations. Examples of such salts include the alkali metal oralkaline-earth metal salts and particularly, the sodium and potassiumsalts. These salts are all prepared by conventional techniques. Thechemical bases which are used as reagents to prepare thepharmaceutically acceptable base salts of this invention are those whichform non-toxic base salts with the acidic compounds of the presentinvention. Such non-toxic base salts include those derived from suchpharmacologically acceptable cations as sodium, potassium calcium andmagnesium, etc. These salts can be prepared by treating thecorresponding acidic compounds with an aqueous solution containing thedesired pharmacologically acceptable cations, and then evaporating theresulting solution to dryness, preferably under reduced pressure.Alternatively, they may also be prepared by mixing lower alkanolicsolutions of the acidic compounds and the desired alkali metal alkoxidetogether, and then evaporating the resulting solution to dryness in thesame manner as before. In either case, stoichiometric quantities ofreagents are preferably employed in order to ensure completeness ofreaction and maximum yields of the desired final product.

If the inventive compound is a base, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method available in theart, for example, treatment of the free base with an inorganic acid,such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, or with an organic acid, such as aceticacid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonicacid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, apyranosidyl acid, such as glucuronic acid or galacturonic acid, analpha-hydroxy acid, such as citric acid or tartaric acid, an amino acid,such as aspartic acid or glutamic acid, an aromatic acid, such asbenzoic acid or cinnamic acid, a sulfonic acid, such asp-toluenesulfonic acid or ethanesulfonic acid, or the like.

If the inventive compound is an acid, the desired pharmaceuticallyacceptable salt may be prepared by any suitable method, for example,treatment of the free acid with an inorganic or organic base, such as anamine (primary, secondary or tertiary), an alkali metal hydroxide oralkaline earth metal hydroxide, or the like. Illustrative examples ofsuitable salts include organic salts derived from amino acids, such asglycine and arginine, ammonia, primary, secondary, and tertiary amines,and cyclic amines, such as piperidine, morpholine and piperazine, andinorganic salts derived from sodium, calcium, potassium, magnesium,manganese, iron, copper, zinc, aluminum and lithium.

In the case of agents that are solids, it is understood by those skilledin the art that the inventive compounds, agents and salts may exist indifferent crystal or polymorphic forms, all of which are intended to bewithin the scope of the present invention and specified formulas.

The HIV Integrase agents of the invention may be formulated intopharmaceutical compositions as described below in any pharmaceuticalform recognizable to the skilled artisan as being suitable.Pharmaceutical compositions of the invention comprise a therapeuticallyeffective amount of at least one compound of Formula I and an inert,pharmaceutically acceptable carrier or diluent.

To treat or prevent diseases or conditions mediated by HIV, apharmaceutical composition of the invention is administered in asuitable formulation prepared by combining a therapeutically effectiveamount (i.e., an HIV Integrase modulating, regulating, or inhibitingamount effective to achieve therapeutic efficacy) of at least onecompound of the present invention (as an active ingredient) with one ormore pharmaceutically suitable carriers, which may be selected, forexample, from diluents, excipients and auxiliaries that facilitateprocessing of the active compounds into the final pharmaceuticalpreparations.

The pharmaceutical carriers employed may be either solid or liquid.Exemplary solid carriers are lactose, sucrose, talc, gelatin, agar,pectin, acacia, magnesium stearate, stearic acid and the like. Exemplaryliquid carriers are syrup, peanut oil, olive oil, water and the like.Similarly, the inventive compositions may include time-delay ortime-release material known in the art, such as glyceryl monostearate orglyceryl distearate alone or with a wax, ethylcellulose,hydroxypropylmethylcellulose, methylmethacrylate or the like. Furtheradditives or excipients may be added to achieve the desired formulationproperties. For example, a bioavailability enhancer, such as Labrasol,Gelucire or the like, or formulator, such as CMC(carboxymethylcellulose), PG (propyleneglycol), or PEG(polyethyleneglycol), may be added. Gelucire®, a semi-solid vehicle thatprotects active ingredients from light, moisture and oxidation, may beadded, e.g., when preparing a capsule formulation.

If a solid carrier is used, the preparation can be tableted, placed in ahard gelatin capsule in powder or pellet form, or formed into a trocheor lozenge. The amount of solid carrier may vary, but generally will befrom about 25 mg to about 1 g. If a liquid carrier is used, thepreparation may be in the form of syrup, emulsion, soft gelatin capsule,sterile injectable solution or suspension in an ampoule or vial ornon-aqueous liquid suspension. If a semi-solid carrier is used, thepreparation may be in the form of hard and soft gelatin capsuleformulations. The inventive compositions are prepared in unit-dosageform appropriate for the mode of administration, e.g., parenteral ororal administration.

To obtain a stable water-soluble dose form, a pharmaceuticallyacceptable salt of a compound of the present invention may be dissolvedin an aqueous solution of an organic or inorganic acid, such as 0.3 Msolution of succinic acid or citric acid. If a soluble salt form is notavailable, the agent may be dissolved in a suitable cosolvent orcombinations of cosolvents. Examples of suitable cosolvents includealcohol, propylene glycol, polyethylene glycol 300, polysorbate 80,glycerin and the like in concentrations ranging from 0–60% of the totalvolume. In an exemplary embodiment, a compound of Formula I is dissolvedin DMSO and diluted with water. The composition may also be in the formof a solution of a salt form of the active ingredient in an appropriateaqueous vehicle such as water or isotonic saline or dextrose solution.

Proper formulation is dependent upon the route of administration chosen.For injection, the agents of the compounds of the present invention maybe formulated into aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks solution, Ringer's solution, orphysiological saline buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carriersknown in the art. Such carriers enable the compounds of the invention tobe formulated as tablets, pills, dragees, capsules, liquids, gels,syrups, slurries, suspensions and the like, for oral ingestion by asubject to be treated. Pharmaceutical preparations for oral use can beobtained using a solid excipient in admixture with the active ingredient(agent), optionally grinding the resulting mixture, and processing themixture of granules after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients include: fillerssuch as sugars, including lactose, sucrose, mannitol, or sorbitol; andcellulose preparations, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as crosslinked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, polyvinyl pyrrolidone, Carbopol gel, polyethylene glycol,and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active agents.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillerssuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate, and, optionally, stabilizers. In softcapsules, the active agents may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration. For buccal administration, the compositions may take theform of tablets or lozenges formulated in conventional manner.

For administration intranasally or by inhalation, the compounds for useaccording to the present invention may be conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof gelatin for use in an inhaler or insufflator and the like may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit-dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active agents may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

In addition to the formulations described above, the compounds of thepresent invention may also be formulated as a depot preparation. Suchlong-acting formulations may be administered by implantation (forexample, subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the compounds may be formulated withsuitable polymeric or hydrophobic materials (for example, as an emulsionin an acceptable oil) or ion-exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

A pharmaceutical carrier for hydrophobic compounds is a cosolvent systemcomprising benzyl alcohol, a nonpolar surfactant, a water-miscibleorganic polymer, and an aqueous phase. The cosolvent system may be a VPDco-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v ofthe nonpolar surfactant polysorbate 80, and 65% w/v polyethylene glycol300, made up to volume in absolute ethanol. The VPD co-solvent system(VPD: 5W) contains VPD diluted 1:1 with a 5% dextrose in water solution.This co-solvent system dissolves hydrophobic compounds well, and itselfproduces low toxicity upon systemic administration. The proportions of aco-solvent system may be suitably varied without destroying itssolubility and toxicity characteristics. Furthermore, the identity ofthe co-solvent components may be varied: for example, other low-toxicitynonpolar surfactants may be used instead of polysorbate 80; the fractionsize of polyethylene glycol may be varied; other biocompatible polymersmay replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and othersugars or polysaccharides may be substituted for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are known examples ofdelivery vehicles or carriers for hydrophobic drugs. Certain organicsolvents such as dimethylsulfoxide also may be employed, althoughusually at the cost of greater toxicity due to the toxic nature of DMSO.Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various sustained-release materialshave been established and are known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

The pharmaceutical compositions also may comprise suitable solid- orgel-phase carriers or excipients. These carriers and excipients mayprovide marked improvement in the bioavailability of poorly solubledrugs. Examples of such carriers or excipients include calciumcarbonate, calcium phosphate, sugars, starches, cellulose derivatives,gelatin, and polymers such as polyethylene glycols. Furthermore,additives or excipients such as Gelucire®, Capryol®, Labrafil®,Labrasol®, Lauroglycol®, Plurol®, Peceol® Transcutol® and the like maybe used. Further, the pharmaceutical composition may be incorporatedinto a skin patch for delivery of the drug directly onto the skin.

It will be appreciated that the actual dosages of the agents of thisinvention will vary according to the particular agent being used, theparticular composition formulated, the mode of administration, and theparticular site, host, and disease being treated. Those skilled in theart using conventional dosage-determination tests in view of theexperimental data for a given compound may ascertain optimal dosages fora given set of conditions. For oral administration, an exemplary dailydose generally employed will be from about 0.001 to about 1000 mg/kg ofbody weight, with courses of treatment repeated at appropriateintervals. Administration of prodrugs may be dosed at weight levels thatare chemically equivalent to the weight levels of the fully activecompounds.

The compounds of the present invention may be administered incombination with an additional agent or agents for the treatment of amammal, such as a human, that is suffering from an infection with theHIV virus, AIDS, AIDS-related complex (ARC), or any other disease orcondition which is related to infection with the HIV virus. The agentsthat may be used in combination with the compounds of the presentinvention include, but are not limited to, those useful as HIV proteaseinhibitors, HIV reverse transcriptase inhibitors, non-nucleoside HIVreverse transcriptase inhibitors, inhibitors of HIV integrase, CCR5inhibitors, HIV fusion inhibitors, compounds useful as immunomodulators,compounds that inhibit the HIV virus by an unknown mechanism, compoundsuseful for the treatment of herpes viruses, compounds useful asanti-infectives, and others as described below.

Compounds useful as HIV protease inhibitors that may be used incombination with the compounds of the present invention include, but arenot limited to, 141 W94 (amprenavir), CGP-73547, CGP-61755, DMP-450,nelfinavir, ritonavir, saquinavir (invirase), lopinavir, TMC-126,BMS-232632 (atazanavir), palinavir, GS-3333, KN 1–413, KNI-272,LG-71350, CGP-61755, PD 173606, PD 177298, PD 178390, PD 178392,U-140690, ABT-378, DMP-450, AG-1776, MK-944, VX-478, indinavir,tipranavir, TMC-114, DPC-681, DPC-684, fosamprenavir calcium (Lexiva),benzenesulfonamide derivatives disclosed in WO 03053435, R-944,Ro-03–34649, VX-385, GS-224338, OPT-TL3, PL-100, SM-309515, AG-148,DG-35-VIII, DMP-850, GW-5950×, KNI-1039, L-756423, LB-71262, LP-130,RS-344, SE-063, UIC-94–003, Vb-19038, A-77003, BMS-182193, BMS-186318,SM-309515, and JE-2147.

Compounds useful as inhibitors of the HIV reverse transcriptase enzymethat may be used in combination with the compounds of the presentinvention include, but are not limited to, abacavir (1592U89), FTC,GS-840, lamivudine (3TC), adefovir dipivoxil, beta-fluoro-ddA, ddC(dideoxycytidine, zalcitabine), ddI (dideoxyinsine, didanosine),stavudine (d4T), zidovudine (AZT), tenofovir, amdoxovir, SPD-754,SPD-756, racivir, reverset (DPC-817), MIV-210 (FLG), beta-L-Fd4C(ACH-126443), MIV-310 (alovudine, FLT), dOTC, DAPD, and emtricitabine.

Compounds useful as non-nucleoside inhibitors of the HIV reversetranscriptase enzyme include, but are not limited to, efavirenz,HBY-097, nevirapine, TMC-120 (dapivirine), TMC-125, delaviradine,DPC-083, DPC-961, TMC-120, capravirine, and tricyclic pyrimidinonederivatives as disclosed in WO 03062238.

Compounds useful as CCR5 inhibitors that may be used in combination withthe compounds of the present invention include, but are not limited to,TAK-779, SC-351125, SCH-D, UK-427857, PRO-140, and GW-873140 (Ono-4128,AK-602).

Compounds useful as inhibitors of HIV integrase enzyme that may be usedin combination with the compounds of the present invention include, butare not limited to, 1,5-naphthyridine-3-carboxamide derivativesdisclosed in WO 03062204, compounds disclosed in WO 03047564, compoundsdisclosed in WO 03049690, and 5-hydroxypyrimidine-4-carboxamidederivatives disclosed in WO 03035076.

Fusion inhibitors for the treatment of HIV that may be used incombination with the compounds of the present invention include, but arenot limited to, T20, T-1249, AMD-3100, and fused tricyclic compoundsdisclosed in JP 2003171381.

Other compounds that are useful inhibitors of HIV that may be used incombination with the compounds of the present invention include, but arenot limited to, Soluble CD4, TNX-355, PRO-542, BMS-806, tenofovirdisoproxil fumarate, and compounds disclosed in JP 2003119137.

Compounds useful in the treatment or management of infection fromviruses other than HIV that may be used in combination with thecompounds of the present invention include, but are not limited to,acyclovir, penciclovir, HPMPC, oxetanocin G, AL-721, cidofovir,cytomegalovirus immune globin, cytovene, ganciclovir, famciclovir, Isis2922, KNI-272, valaciclovir, and virazole ribavirin.

Compounds that act as immunomodulators and may be used in combinationwith the compounds of the present invention include, but are not limitedto, AD-439, AD-519, Alpha Interferon, AS-101, bropirimine, acemannan,CL246,738, EL10, FP-21399, gamma interferon, granulocyte macrophagecolony stimulating factor, IL-2, immune globulin intravenous, IMREG-1,IMREG-2, imuthiol diethyl dithio carbamate, alpha-2 interferon,methionine-enkephalin, MTP-PE, granulocyte colony stimulating sactor,remune, rCD4, recombinant soluble human CD4, interferon alfa-2,SK&F106528, soluble T4 yhymopentin, tumor necrosis factor (TNF),tucaresol, recombinant human interferon beta, and interferon alfa n-3.

Anti-infectives that may be used in combination with the compounds ofthe present invention include, but are not limited to, clindamycin withprimaquine, fluconazole, pastill, ornidyl, eflornithine pentamidine,spiramycin, intraconazole-R51211, trimetrexate, daunorubicin,recombinant human erythropoietin, recombinant human growth hormone,megestrol acetate, testerone, and total enteral nutrition.

Other compounds that may be used in combination with the compounds ofthe present invention include, but are not limited to, acmannan,ansamycin, LM 427, AR177, BMS-232623, BMS-234475, CI-1012, curdlansulfate, dextran sulfate, STOCRINE EL10, hypericin, lobucavir, novapren,peptide T octabpeptide sequence, trisodium phosphonoformate, probucol,and RBC-CD4.

In addition, the compounds of the present invention may be used incombination with compounds that act as inhibitors of metallo-matrixproteases, so-called MMP inhibitors.

The particular choice of an additional agent or agents will depend on anumber of factors that include, but are not limited to, the condition ofthe mammal being treated, the particular condition or conditions beingtreated, the identity of the compound or compounds of the presentinvention and the additional agent or agents, and the identity of anyadditional compounds that are being used to treat the mammal. Theparticular choice of the compound or compounds of the invention and theadditional agent or agents is within the knowledge of one of ordinaryskill in the art.

The compounds of the present invention may be administered incombination with any of the above additional agents for the treatment ofa mammal, such as a human, that is suffering from an infection with theHIV virus, AIDS, AIDS-related complex (ARC), or any other disease orcondition which is related to infection with the HIV virus. Such acombination may be administered to a mammal such that a compound orcompounds of the present invention are present in the same formulationas the additional agents described above. Alternatively, such acombination may be administered to a mammal suffering from infectionwith the HIV virus such that the compound or compounds of the presentinvention are present in a formulation that is separate from theformulation in which the additional agent is found. If the compound orcompounds of the present invention are administered separately from theadditional agent, such administration may take place concomitantly orsequentially with an appropriate period of time in between. The choiceof whether to include the compound or compounds of the present inventionin the same formulation as the additional agent or agents is within theknowledge of one of ordinary skill in the art.

Additionally, the compounds of the present invention may be administeredto a mammal, such as a human, in combination with an additional agentthat has the effect of increasing the exposure of the mammal to acompound of the invention. The term “exposure,” as used herein, refersto the concentration of a compound of the invention in the plasma of amammal as measured over a period of time. The exposure of a mammal to aparticular compound can be measured by administering a compound of theinvention to a mammal in an appropriate form, withdrawing plasma samplesat predetermined times, and measuring the amount of a compound of theinvention in the plasma using an appropriate analytical technique, suchas liquid chromatography or liquid chromatography/mass spectroscopy. Theamount of a compound of the invention present in the plasma at a certaintime is determined and the concentration and time data from all thesamples are plotted to afford a curve. The area under this curve iscalculated and affords the exposure of the mammal to the compound. Theterms “exposure,” “area under the curve,” and “area under theconcentration/time curve” are intended to have the same meaning and maybe used interchangeably throughout.

Among the agents that may be used to increase the exposure of a mammalto a compound of the present invention are those that can as inhibitorsof at least one isoform of the cytochrome P450 (CYP450) enzymes. Theisoforms of CYP450 that may be beneficially inhibited include, but arenot limited to, CYP1A2, CYP2D6, CYP2C9, CYP2C19 and CYP3A4. Suitableagents that may be used to inhibit CYP 3A4 include, but are not limitedto, ritonavir.

Such a combination may be administered to a mammal such that a compoundor compounds of the present invention are present in the sameformulation as the additional agents described above. Alternatively,such a combination may be administered such that the compound orcompounds of the present invention are present in a formulation that isseparate from the formulation in which the additional agent is found. Ifthe compound or compounds of the present invention are administeredseparately from the additional agent, such administration may take placeconcomitantly or sequentially with an appropriate period of time inbetween. The choice of whether to include the compound or compounds ofthe present invention in the same formulation as the additional agent oragents is within the knowledge of one of ordinary skill in the art.

Several different assay formats are available to measureintegrase-mediated integration of viral DNA into target (or host) DNAand thus, identify compounds that modulate (e.g., inhibit) integraseactivity. In general, for example, ligand-binding assays may be used todetermine interaction with an enzyme of interest. When binding is ofinterest, a labeled enzyme may be used, wherein the label is afluorescer, radioisotope, or the like, which registers a quantifiablechange upon binding to the enzyme. Alternatively, the skilled artisanmay employ an antibody for binding to the enzyme, wherein the antibodyis labeled allowing for amplification of the signal. Thus, binding maybe determined through direct measurement of ligand binding to an enzyme.In addition, binding may be determined by competitive displacement of aligand bound to an enzyme, wherein the ligand is labeled with adetectable label. When inhibitory activity is of interest, an intactorganism or cell may be studied, and the change in an organismic orcellular function in response to the binding of the inhibitory compoundmay be measured. Alternatively, cellular response can be determinedmicroscopically by monitoring viral induced syncytium-formation (HIV-1syncytium-formation assays), for example. Thus, there are various invitro and in vivo assays useful for measuring HIV integrase inhibitoryactivity. See, e.g., Lewin, S. R. et al., Journal of Virology 73(7):6099–6103 (July 1999); Hansen, M. S. et al., Nature Biotechnology 17(6):578–582 (June 1999); and Butler, S. L. et al., Nature Medicine 7(5):631–634 (May 2001).

Exemplary specific assay formats used to measure integrase-mediatedintegration include, but are not limited to, ELISA, DELFIA® (PerkinElmerLife Sciences Inc. (Boston, Mass.)) and ORIGEN® (IGEN International,Inc. (Gaithersburg, Md.)) technologies. In addition, gel-basedintegration (detecting integration by measuring product formation withSDS-PAGE) and scintillation proximity assay (SPA) disintegration assaysthat use a single unit of double stranded-DNA (ds-DNA) may be used tomonitor integrase activity.

In one embodiment of the invention, the preferred assay is an integrasestrand-transfer SPA (stINTSPA) which uses SPA to specifically measurethe strand-transfer mechanism of integrase in a homogenous assayscalable for miniaturization to allow high-throughput screening. Theassay focuses on strand transfer and not on DNA binding and/or 3′processing. This sensitive and reproducible assay is capable ofdistinguishing non-specific interactions from true enzymatic function byforming 3′ processed viral DNA/integrase complexes before the additionof target DNA. Such a formation creates a bias toward compoundmodulators (e.g., inhibitors) of strand-transfer and not towardcompounds that inhibit integrase 3′ processing or prevent theassociation of integrase with viral DNA. This bias renders the assaymore specific than known assays. In addition, the homogenous nature ofthe assay reduces the number of steps required to run the assay sincethe wash steps of a heterogenous assay are not required.

The integrase strand-transfer SPA format consists of 2 DNA componentsthat model viral DNA and target DNA. The model viral DNA (also known asdonor DNA) is biotinylated ds-DNA preprocessed at the 3′ end to providea CA nucleotide base overhang at the 5′ end of the duplex. The targetDNA (also known as host DNA) is a random nucleotide sequence of ds-DNAgenerally containing [³H]-thymidine nucleotides on both strands,preferably, at the 3′ ends, to enable detection of the integrasestrand-transfer reaction that occurs on both strands of target ds-DNA.

Integrase (created recombinantly or synthetically and preferably,purified) is pre-complexed to the viral DNA bound to a surface, such asfor example, streptavidin-coated SPA beads. Generally, the integrase ispre-complexed in a batch process by combining and incubating dilutedviral DNA with integrase and then removing unbound integrase. Thepreferred molar ratio of viral DNA:integrase is about 1:about 5. Theintegrase/viral DNA incubation is optional, however, the incubation doesprovide for an increased specificity index with an integrase/viral DNAincubation time of about 15 to about 30 minutes at room temperature orat about 37° C. The preferred incubation is at about room temperaturefor about 15 minutes.

The reaction is initiated by adding target DNA, in the absence orpresence of a potential integrase modulator compound, to theintegrase/viral DNA beads (for example) and allowed to run for about 20to about 50 minutes (depending on the type of assay container employed),at about room temperature or about 37° C., preferably, at about 37° C.The assay is terminated by adding stop buffer to the integrase reactionmixture. Components of the stop buffer, added sequentially or at onetime, function to terminate enzymatic activity, dissociate integrase/DNAcomplexes, separate non-integrated DNA strands (denaturation agent),and, optionally, float the SPA beads to the surface of the reactionmixture to be closer in range to the detectors of, for example, aplate-based scintillation counter, to measure the level of integratedviral DNA which is quantified as light emitted (radiolabeled signal)from the SPA beads. The inclusion of an additional component in the stopbuffer, such as for example CsCl or functionally equivalent compound, isoptionally, and preferably, used with a plate-based scintillationcounter, for example, with detectors positioned above the assay wells,such as for example a TopCount® counter (PerkinElmer Life Sciences Inc.(Boston, Mass.)). CsCl would not be employed when PMT readings are takenfrom the bottom of the plate, such as for example when a MicroBeta®counter (PerkinElmer Life Sciences Inc. (Boston, Mass.)) is used.

The specificity of the reaction can be determined from the ratio of thesignal generated from the target DNA reaction with the viralDNA/integrase compared to the signal generated from the di-deoxy viralDNA/integrase. High concentrations (e.g., ≧50 nM) of target DNA mayincrease the d/dd DNA ratio along with an increased concentration ofintegrase in the integrase/viral DNA sample.

The results can be used to evaluate the integrase modulatory, such asfor example inhibitory, activity of test compounds. For example, theskilled artisan may employ a high-throughput screening method to testcombinatorial compound libraries or synthetic compounds. The percentinhibition of the compound may be calculated using an equation such asfor example (1−((CPM sample−CPM min)/(CPM max−CPM min)))*100. The minvalue is the assay signal in the presence of a known modulator, such asfor example an inhibitor, at a concentration about 100-fold higher thanthe IC₅₀ for that compound. The min signal approximates the truebackground for the assay. The max value is the assay signal obtained forthe integrase-mediated activity in the absence of compound. In addition,the IC₅₀ values of synthetic and purified combinatorial compounds may bedetermined whereby compounds are prepared at about 10 or 100-fold higherconcentrations than desired for testing in assays, followed by dilutionof the compounds to generate an 8-point titration curve with ½-logdilution intervals, for example. The compound sample is then transferredto an assay well, for example. Further dilutions, such as for example, a10-fold dilution, are optional. The percentage inhibition for aninhibitory compound, for example, may then be determined as above withvalues applied to a nonlinear regression, sigmoidal dose responseequation (variable slope) using GraphPad Prism curve fitting software(GraphPad Software, Inc., San Diego, Calif.) or functionally equivalentsoftware.

The stINTSPA assay conditions are preferably optimized for ratios ofintegrase, viral DNA and target DNA to generate a large and specificassay signal. A specific assay signal is defined as a signaldistinguishing true strand-transfer catalytic events from complexformation of integrase and DNA that does not yield product. In otherintegrase assays, a large non-specific component (background) oftencontributes to the total assay signal unless the buffer conditions arerigorously optimized and counter-tested using a modified viral DNAoligonucleotide. The non-specific background is due to formation ofintegrase/viral DNA/target DNA complexes that are highly stableindependent of a productive strand-transfer mechanism.

The preferred stINTSPA distinguishes complex formation from productivestrand-transfer reactions by using a modified viral DNA oligonucleotidecontaining a di-deoxy nucleoside at the 3′ end as a control. Thismodified control DNA can be incorporated into integrase/viral DNA/targetDNA complexes, but cannot serve as a substrate for strand-transfer.Thus, a distinct window between productive and non-productivestrand-transfer reactions can be observed. Further, reactions withdi-deoxy viral DNA beads give an assay signal closely matched to thetrue background of the assay using the preferred optimization conditionsof the assay. The true background of the assay is defined as a reactionwith all assay components (viral DNA and [³H]-target DNA) in the absenceof integrase.

Assay buffers used in the integrase assay generally contain at least onereducing agent, such as for example 2-mercaptoethanol or DTT, whereinDTT as a fresh powder is preferred; at least one divalent cation, suchas for example Mg⁺⁺, Mn⁺⁺, or Zn⁺⁺, preferably, Mg⁺⁺; at least oneemulsifier/dispersing agent, such as for example octoxynol (also knownas IGEPAL-CA or NP-40) or CHAPS; NaCl or functionally equivalentcompound; DMSO or functionally equivalent compound; and at least onebuffer, such as for example MOPS. Key buffer characteristics are theabsence of PEG; inclusion of a high concentration of a detergent, suchas for example about 1 to about 5 mM CHAPS and/or about 0.02 to about0.15% IGEPAL-CA or functionally equivalent compound(s) at least capableof reducing non-specific sticking to the SPA beads and assay wells and,possibly, enhancing the specificity index; inclusion of a highconcentration of DMSO (about 1 to about 12%); and inclusion of modestlevels of NaCl (≦50 mM) and MgCl₂ (about 3 to about 10 mM) orfunctionally equivalent compounds capable of reducing the dd-DNAbackground. The assay buffers may optionally contain a preservative,such as for example NaN₃, to reduce fungal and bacterial contaminantsduring storage.

The stop buffer preferably contains EDTA or functionally equivalentcompound capable of terminating enzymatic activity, a denaturation agentcomprising, for example, NaOH or guanidine hydrochloride, and,optionally, CsCl or functionally equivalent compound capable ofassisting in floating the SPA beads to the top of the assay containerfor scintillation detection at the top of the reservoir and, possibly,minimizing compound interference. An example of an integrasestrand-transfer SPA is set forth in Example 13.

Alternatively, the level of activity of the modulatory compounds may bedetermined in an antiviral assay, such as for example an assay thatquantitatively measures the production of viral antigens (e.g., HIV-1p24) or the activities of viral enzymes (e.g., HIV-1 reversetranscriptase) as indicators of virus replication, or that measuresviral replication by monitoring the expression of an exogenous reportergene introduced into the viral genome (HIV-1 reporter virus assays)(Chen, B. K. et al., J. Virol. 68(2): 654–660 (1994); Terwilliger, E. F.et al., PNAS 86: 3857–3861 (1989)). A preferred method of measuringantiviral activity of a potential modulator compound employs an HIV-1cell protection assay, wherein virus replication is measured indirectlyby monitoring viral induced host-cell cytopathic effects using, forexample, dye reduction methods as set forth in Example 14.

In one embodiment, the compounds of the present invention include thosehaving an EC₅₀ value against HIV integrase of at least 10⁻⁵ M (or atleast 10 μM) when measured with an HIV cell protection assay. In anotherembodiment are compounds of the present invention with an EC₅₀ valueagainst HIV integrase of at least 1 μM when measured with an HIV cellprotection assay. In yet another embodiment, the compounds of thepresent invention have an EC₅₀ against HIV integrase of at least 0.1 μMwhen measured with an HIV cell protection assay.

The inventive agents may be prepared using the reaction routes andsynthesis schemes as described below, employing the techniques availablein the art using starting materials that are readily available. Thepreparation of preferred compounds of the present invention is describedin detail in the following examples, but the artisan will recognize thatthe chemical reactions described may be readily adapted to prepare anumber of other HIV Integrase agents of the invention. For example, thesynthesis of non-exemplified compounds according to the invention may beperformed by modifications apparent to those skilled in the art, e.g.,by appropriately protecting interfering groups, by changing to othersuitable reagents known in the art, or by making routine modificationsof reaction conditions. Alternatively, other reactions disclosed hereinor known in the art will be recognized as having adaptability forpreparing other compounds of the invention.

Reagents useful for synthesizing compounds may be obtained or preparedaccording to techniques known in the art. For example, the preparationof free amines from common salt forms and stock reagent solutions can beuseful for small-scale reactions. See also Abdel-Magid et al.,“Reductive Amination of Aldehydes and Ketones with SodiumTriacetoxyborohydride,” J. Org. Chem. 61: 3849 (1996).

Methanolic solutions of the free bases can be prepared fromhydrochloride, dihydrochloride, hydrobromide, or other salts when thefree base is soluble in methanol. In this procedure, once the sodiummethoxide is added, care should be taken to prevent exposure to air,since amine free bases, particularly primary amines, absorb carbondioxide from the air to form salts. A 10-mL quantity of a 0.1M solutionof a free base in methanol may be prepared as follows. Weigh 1.0 mmol ofa monohydrochloride salt into a tared Erlenmeyer flask containing astirring bar, and add 7 mL of methanol. To the stirred slurry, add 229mL (1.0 mmol, 1 equiv.) of sodium methoxide in methanol (25 wt %, 4.37M), stopper the flask, and stir the mixture vigorously for 2 hours. Theslurry will sometimes change in appearance as a finer, milky precipitateof sodium chloride is formed. Filter the slurry through a 15-mL mediumfritted glass funnel, wash the filter case with 1–2 mL methanol,transfer the filtrate to a 20-mL vial, and dilute to 10 mL withmethanol. The theoretical yield of sodium chloride is nearly 59 mg, butthe recovery is usually not quantitative, owing to a slight solubilityin methanol. For a dihydrochloride salt, a second equivalent of sodiummethoxide is required (458 mL).

A 0.5 M solution of sodium borohydride in ethanol may be prepared asfollows. Sodium borohydride (520 mg, 13.8 mmol) is stirred in pure(non-denatured) anhydrous ethanol (25 mL) for ˜2–3 minutes. Thesuspension is filtered through a medium fritted glass funnel to remove asmall amount of undissolved solid (typically about 5% of the total massof borohydride, or 25 mg). The filtrate should appear as a colorlesssolution that evolves only a little hydrogen. This solution should beused immediately, as it decomposes significantly over a period of a fewhours, resulting in the formation of a gelatinous precipitate. Sodiumborohydride is hygroscopic, so avoid exposure to air by making thesolution at once after weighing the solid. Sodium borohydride has asolubility of about 4% in ethanol at room temperature. This correspondsto a little over 0.8 M. However, sometimes a small percentage of thesolid remains undissolved regardless of the concentration beingprepared, even after stirring for ≧5 minutes.

The following abbreviations employed throughout the application have thefollowing meaning unless otherwise indicated:

-   NaH: sodium hydride;-   THF: tetrahydrofuran;-   DMF: N,N-dimethylformamide;-   TLC: thin-layer-chromatography;-   HATU: O-(7-azabenzotriazole-1-yl)-N,N, N′,N′-tetramethyl uronium    hexafluorophosphate;-   EDC: N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide;-   DMSO: dimethyl sulfoxide;-   DDQ: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone;-   MS: molecular sieve(s);-   NaBH₃CN: sodium cyanoborohydride;-   TiCl₄: titanium (IV) tetrachloride;-   AcOH: acetic acid;-   TFA: trifluoro acetic acid;-   PPTS: pyridinium p-toluenesulfonate; and-   HOBt: 1-hydroxybenzotriazole    Additional abbreviations employed throughout the application are    either known to those skilled in the art or are explained in the    Examples below.

EXAMPLES

The present invention will be further illustrated in the following,non-limiting examples. In the examples described below, unless otherwiseindicated, all temperatures in the following description are in degreesCelsius and all parts and percentages are by weight, unless indicatedotherwise.

Various starting materials and other reagents were purchased fromcommercial suppliers, such as Aldrich Chemical Company or LancasterSynthesis Ltd., and used without further purification, unless otherwiseindicated. Tetrahydrofuran (THF) and N,N-dimethylformamide (DMF) werepurchased from Aldrich in SureSeal® bottles and used as received. Allsolvents were purified using methods standard in the art, unlessotherwise indicated.

The reactions set forth below were performed under a positive pressureof nitrogen, argon or with a drying tube, at ambient temperature (unlessotherwise stated), in anhydrous solvents, and the reaction flasks arefitted with rubber septa for the introduction of substrates and reagentsvia syringe. Glassware was oven-dried and/or heat-dried. Analyticalthin-layer chromatography was performed on glass-backed silica gel 60°F. 254 plates (Analtech (0.25 mm)) and eluted with the appropriatesolvent ratios (v/v). The reactions were assayed by TLC and terminatedas judged by the consumption of starting material.

The TLC plates were visualized by UV absorption or with a p-anisaldehydespray reagent or a phosphomolybdic acid reagent (Aldrich Chemical, 20 wt% in ethanol) which was activated with heat. Work-ups were typicallydone by doubling the reaction volume with the reaction solvent orextraction solvent and then washing with the indicated aqueous solutionsusing 25% by volume of the extraction volume (unless otherwiseindicated). Product solutions were dried over anhydrous Na₂SO₄ prior tofiltration, and evaporation of the solvents was under reduced pressureon a rotary evaporator and noted as solvents removed in vacuo. Flashcolumn chromatography [Still et al., A. J. Org. Chem. 43: 2923 (1978)]was conducted using Baker-grade flash silica gel (47–61 mm) and a silicagel: crude material ratio of about 20:1 to 50:1, unless otherwisestated. Hydrogenolysis was done at the pressure indicated or at ambientpressure.

¹H-NMR spectra were recorded on a Bruker instrument operating at 300MHz, 500 MHz, and ¹³C-NMR spectra was recorded operating at 75 MHz. NMRspectra are obtained as CDCl₃ solutions (reported in ppm), usingchloroform as the reference standard (7.25 ppm and 77.00 ppm) or CD₃OD(3.4 and 4.8 ppm and 49.3 ppm), or an internal tetramethylsilanestandard (0.00 ppm) when appropriate. Other NMR solvents were used asneeded. When peak multiplicities are reported, the followingabbreviations are used: s=singlet, d=doublet, t=triplet, m=multiplet,br=broadened, dd=doublet of doublets, dt=doublet of triplets. Couplingconstants, when given, are reported in Hertz.

Infrared spectra were recorded on a Perkin-Elmer FT-IR Spectrometer asneat oils, as KBr pellets, or as CDCl₃ solutions, and when reported arein wave numbers (cm⁻¹). The mass spectra were obtained using LC/MS orAPCI. All melting points are uncorrected.

All final products had greater than 85% purity (by HPLC at wavelengthsof 220 nm and 254 nm).

General Procedures

Scheme 1 represents a method for preparing compounds 1-4 of the presentinvention directly from ester 1-2 (where R is typically a methyl orethyl and R₁–R₉ are as defined above) and a substituted or unsubstitutedhydroxylamine, in the absence or presence of a base such as sodiumhydroxide in methanol or ethanol (C. R. Hauser, et al., Org. Synth.Coll., Vol. 2, p. 67, John Wiley, New York (1943)). The ester 1-2 can bemade by alkylation of compound 1-1 with R₇X in the presence of NaH inDMF or DMSO (M. K. Eberle, J. Org. Chem., 41, 633 (1976); R. J.Sundberg, et al., J. Org. Chem., 38, 3324). Alternatively, the ester 1-2can be saponified to the free acid 1-3, which can then be coupled with asubstituted or unsubstituted hydroxylamine using a coupling reagent,such as O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyl uroniumhexafluorophosphate (HATU) orN-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC), or many others thatare familiar to those skilled in the art, to give the compounds 1-4 ofthe present invention. Suitable methods are described, for example, inJerry March, Advanced Organic Chemistry, 5th edition, pp. 508–511, JohnWhiley & Sons (2001). Use of the conditions as set forth in the Examplesbelow allows for the parallel preparation or combinatorial syntheses ofhydroxamates 1-4.

Preparation of Intermediates and Starting Materials

Scheme 2 represents a convenient method for preparation of substitutedβ-carboline compounds 1-1 (where R=ethyl and R₂–R₆ are as definedabove)(G. Neef, et al., Heterocycles, 20, 1295 (1983)). The synthesisemploys standard methods of indole chemistry: aldimines 2-2 can beprepared by Campbell's procedure (K. N. Campbell, et al., J. Am. Chem.Soc., 66, 82 (1944)) and reacted with indoles in analogy to Snyder'sreaction mode (H. R. Snyder, et al., J. Am. Chem. Soc., 79, 2217 (1957))to give compounds 2-3. Condensation of compounds 2-3 with ethyl nitroacetate can be performed as described by Lyttle and Erofeev (D. A.Lyttle, et al., J. Am. Chem. Soc., 69, 2118 (1947); Y. V. Erofeev, etal., Khim. Get. Soed., 780 (1978)) to yield nitro compounds 2-4.Compounds 2-4 can also be conveniently obtained by Michael type additionof indoles to the nitro ester 2-5 (R₂—CH═C(NO₂)—COOEt) prepared bycondensation of aldehydes (R₂CHO) with ethyl nitro acetate(O₂NCH₂COOEt). Hydrogenation of compounds 2-4 in the presence ofRaney-Ni can give tryptophan derivatives 2-6, as mixtures of isomers.Pictet-Spengler reaction using Sandrin's modification (J. Sandrin etal., Heterocycles, 4, 1101 (1976)) can produce tetrahydro-β-carbolines2-7, which without further purification, can be subjected to oxidationby DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone), sulfur or palladiumto afford the desired ester compounds 1-1.

Scheme 3 describes another efficient route to obtain β-carboline esters1-1 (where R=ethyl or methyl; R₁=H; and R₂–R₆ are as defined above) (M.Dekhane, et al., Tetrahedron, 50, 6299 (1994)). Condensation ofaldehydes 3-4 and amino ketal 3-3 (prepared by the method of Belleau, T.W. Doyle et al., Can. J. Chem., 55, 468 (1977)) in the presence of 4 Åmolecular sieves (MS) can yield imines 3-5, which can be reduced toamines 3-6 with sodium cyanoborohydride (NaBH₃CN) in ethanol. Ontreatment of the amines 3-6 with titanium (IV) tetrachloride (TiCl₄) thedesired β-carboline esters 1-1 can be obtained.

β-Carboline esters 1-1 (where R=methyl or ethyl; R₁, R₂=H; and R₃–R₆ areas defined above) can also be prepared by the one pot procedure outlinedin Scheme 4. Indole derivatives 4-1 can react with 2-azabutadienederivatives 4-2 (where R=methyl or ethyl) (W. Kantlehner, et al.,Liebigs Ann. Chem., 344 (1980)) in the presence of acetic acid (AcOH)and trifluoro acetic acid (TFA) following the procedures described in H.Biere at al., Liebigs Ann. Chem., 1749 (1986) to form substitutedβ-carboline esters 1-1 directly.

Scheme 5 represents another method for obtaining substituted β-carbolineesters 1-1 (R=methyl or ethyl; R₂=H; and R₁ and R₃–R₆ are as definedabove). Refluxing of tryptophan derivatives 5-1 with aryl aldehyde 5-2(R₁=aryl) in the presence of pyridinium p-toluenesulfonate (PPTS) intoluene can afford the desired β-carboline esters 1-1 in one step (C.Barbier et al., Heterocycles, 1, 37 (2000)).

Other compounds of the Formula I may be prepared in manners analogous tothe general procedures described above or the detailed proceduresdescribed in the following examples:

Example 1 9-(4-Fluorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide

To a stirred solution of ethyl 9H-β-carboline-3-carboxylate (368 mg,1.53 mmol) in DMF (4 mL) under a nitrogen atmosphere was added NaH (61.2mg, 60% in mineral oil, 1.53 mmol) portionwise, followed by4-fluorobenzyl bromide (0.19 mL, 1.53 mmol). Stirring was continued for24 hours at ambient temperature, water (10 mL) was then added to themixture. The precipitate was filtered, washed with water and dried togive a solid that was dissolved in methanol (20 mL). To the resultingsolution, H₂NOH (20 mL, 50 wt. % solution in H₂O, 0.30 mol) was added.The suspension was stirred for 5 days at ambient temperature. Themixture was then filtered, and the solid was boiled in methanol (40 mL).After filtration, the title product (0.20 g, 39%) was obtained. ¹H NMR(300 MHz, DMSO-d₆): δ 11.28 (1H, s), 9.02–9.08 (2H, m), 8.85 (1H, s),8.48 (1H, d, J=9.0 Hz), 7.10–7.84 (7H, m), 5.85 (2H, s). HRMS (M+H)⁺found: 336.1157. Calcd for C₁₉H₁₅N₃O₂F: 336.1148.

Example 29-[(5-Chlorothien-2-yl)methyl]-N-hydroxy-9H-β-carboline-3-carboxamide

To a stirred solution of ethyl 9Hβ-carboline-3-carboxylate (300 mg, 1.25mmol) in DMF (3 mL) under a nitrogen atmosphere was added NaH (50.0 mg,60% in mineral oil, 1.25 mmol) portionwise, followed by2-chloro-5-(chloromethyl)-thiophene (151 μL, 1.25 mmol). The stirringwas continued for 21 hours at ambient temperature, water (50 mL) wasthen added to the mixture. The precipitate was filtered, washed withwater and dried to give a solid that was dissolved in methanol (25 mL).To the resulting solution, H₂NOH (25 mL, 50 wt. % solution in H₂O, 0.38mol) was added. The suspension was stirred for 4 days at ambienttemperature. The mixture was then filtered, and the solid was boiled inmethanol (25 mL). After filtration, the desired product (0.26 g, 58%)was obtained. ¹H NMR (400 MHz, DMSO-d₆): □ 11.28 (1H, s), 9.15 (1H, s),9.01 (1H, s), 8.81 (1H, s), 8.44 (1H, d, J=8 Hz), 6.94–7.92 (5H, m),5.99 (2H, s). HRMS (M+H)⁺ found: 358.0417. Calcd for C₁₇H₁₃N₃O₂SCl:358.0417.

Example 39-(3-Chloro-2-fluorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide

To a stirred solution of ethyl 9H-β-carboline-3-carboxylate (400 mg,1.66 mmol) in DMF (5 mL) under nitrogen atmosphere was added NaH (66.4mg, 60% in mineral oil, 1.66 mmol) portionwise, followed by3-chloro-2-fluoro-benzyl bromide (371 mg, 1.66 mmol). Stirring wascontinued for 25 hours at ambient temperature, water (30 mL) was thenadded to the mixture. The precipitate was filtered, washed with waterand dried to give a solid that was dissolved in methanol (25 mL). To theresulting solution, H₂NOH (25 mL, 50 wt. % solution in H₂O, 0.38 mol)was added. The suspension was stirred for 4 days at ambient temperature.The mixture was then filtered, and the solid was boiled in methanol (20mL). After filtration, the title product (0.19 g, 31%) was obtained. ¹HNMR (400 MHz, DMSO-d₆): δ 11.33 (1H, s), 9.08 (1H, s), 9.04 (1H, s),8.89 (1H, s), 8.51 (1H, d, J=8 Hz), 6.95–7.81 (6H, m), 6.00 (2H, s).HRMS (M+H)⁺ found: 370.0764. Calcd for C₁₉H₁₄N₃O₂FCl: 370.0759.

Example 4 9-Benzyl-N-hydroxy-9H-β-carboline-3-carboxamide

To a stirred solution of ethyl 9H-β-carboline-3-carboxylate (290 mg,1.21 mmol) in DMF (4 mL) under a nitrogen atmosphere was added NaH (53mg, 60% in mineral oil, 1.33 mmol) portionwise, followed by benzylbromide (173 μL, 1.45 mmol). Stirring was continued for 21 hours atambient temperature, water (50 mL) was then added to the mixture. Theprecipitate was filtered, washed with water and dried to give a solidthat was dissolved in methanol (25 mL). To the resulting solution, H₂NOH(25 mL, 50 wt. % solution in H₂O, 0.38 mol) was added. The suspensionwas stirred for 3 days at ambient temperature. The mixture was thenfiltered, and the solid was boiled in methanol (25 mL). Afterfiltration, the title product (0.15 g, 39%) was obtained. ¹H NMR (400MHz, DMSO-d₆): δ 11.32 (1H, s), 9.10 (1H, s), 9.05 (1H, s), 8.89 (1H,s), 8.52 (1H, d, J=8 Hz), 7.26–7.87 (8H, m), 5.90 (2H, s). HRMS (M+H)⁺found: 318.1249. Calcd for C₁₉H₁₆N₃O₂: 318.1243.

Example 5 9-(4-Methylbenzyl)-N-Hydroxy-9H-β-carboline-3-carboxamide

To a stirred solution of ethyl 9H-β-carboline-3-carboxylate (400 mg,1.67 mmol) in DMF (5 mL) under a nitrogen atmosphere was added NaH (73mg, 60% in mineral oil, 1.76 mmol) portionwise, followed by4-methylbenzyl bromide (308 mg, 1.67 mmol). Stirring was continued for48 hours at ambient temperature, water (50 mL) was then added to themixture. The precipitate was filtered, washed with water and dried togive a solid that was dissolved in methanol (35 mL). To the resultingsolution, NaOH (2N, 1 mL) and H₂NOH (35 mL, 50 wt. % solution in H₂O,0.53 mol) were added. The suspension was stirred for 7 days at ambienttemperature. The mixture was then filtered and dried to the titleproduct (0.20 g, 36%). ¹H NMR (400 MHz, DMSO-d₆): δ 11.25 (1H, s),8.95–9.03 (2H, m), 8.83 (1H, s), 8.45 (1H, d, J=8 Hz), 7.05–7.81 (7H,m), 5.78 (2H, s), 2.21 (3H, s). HRMS (M+H)⁺ found: 332.1400. Calcd forC₂₀H₁₈N₃O₂: 332.1399.

Example 6 9-(2,4-Difluorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide

To a stirred solution of ethyl 9H-β-carboline-3-carboxylate (370 mg,1.54 mmol) in DMF (5 mL) under a nitrogen atmosphere was added NaH (61.6mg, 60% in mineral oil, 1.54 mmol) portionwise, followed by2,4-difluorobenzyl bromide (198 μL, 1.54 mmol). Stirring was continuedfor 48 hours at ambient temperature, water (100 mL) was then added tothe mixture. The precipitate was filtered, washed with water and driedto give a solid that was dissolved in methanol (35 mL). To the resultingsolution, H₂NOH (30 mL, 50 wt. % solution in H₂O, 0.45 mol) was added.The suspension was stirred for 4 days at ambient temperature. Themixture was then filtered, and the solid was recrystallized frommethanol to give the title product (0.20 g, 37%). ¹H NMR (400 MHz,DMSO-d₆): δ 11.46 (1H, s), 9.15–9.22 (2H, m), 9.02 (1H, s), 8.64 (1H, d,J=8 Hz), 7.15–7.96 (6H, m), 6.06 (2H, s). HRMS (M+H)⁺ found: 354.1062.Calcd for C₁₉H₁₄F₂N₃O₂FCl: 354.1054.

Example 79-(3-Chloro-2,6-difluorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide

Step (a): Ethyl9-(3-chloro-2,6-difluorobenzyl)-9H-β-carboline-3-carboxylate. To astirred solution of ethyl 9H-β-carboline-3-carboxylate (440 mg, 1.83mmol) in DMF (5 mL) under a nitrogen atmosphere was added NaH (73.3 mg,60% in mineral oil, 1.83 mmol) portionwise, followed by3-chloro-2,6-difluorobenzyl bromide (442 mg, 1.83 mmol). Stirring wascontinued for 22 hours at ambient temperature, water (30 mL) was thenadded to the mixture. The precipitate was filtered, washed with waterand dried to give the title product (0.55 g, 68%). ¹H NMR (400 MHz,CD₃OD): δ 9.04 (1H, s), 8.86 (1H, s), 8.26 (1H, d, J=8 Hz), 6.98–7.69(5H, m), 5.82 (2H, s), 4.44 (2H, q, J=8 Hz), 1.43 (3H, t, J=8 Hz).

Step (b):9-(3-Chloro-2,6-difluorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide.To a stirred solution of ethyl9-(3-chloro-2,6-difluorobenzyl)-9H-β-carboline-3-carboxylate (260 mg,0.649 mmol) in methanol (25 mL) under a nitrogen atmosphere was addedLiOH (4 mL, 1N in H₂O, 4.0 mmol). The stirring was continued for 24hours at ambient temperature, 20% of citric acid was then added toneutralize the mixture. The solvents were removed under reducedpressure. H₂O (20 mL) was finally added to the resulting residue, andthe precipitates were collected, washed with water and dried to give thecorresponding acid (169 mg, 70%) that was directly used for the nextstep.

To the stirred solution of the acid (169 mg, 0.45 mmol) in DMF (8 mL)were added 1-[3-(dimethylamino)propyl]-3-ethyl-carbodiimidehydrochloride (EDC, 234 mg, 1.22 mmol) and HOBt (142 mg, 1.05 mmol). Themixture was stirred for 1 h, and then triethylamine (0.62 ml, 4.4 mmol)and hydroxylamine hydrochloride (243 mg, 3.49 mmol) were added. Theresulting mixture was stirred for 48 h at ambient temperature, and thenwater (100 mL) was added. The precipitates were collected, washed withmethanol, and dried to give the title compound (130 mg, 74%). ¹H NMR(400 MHz, DMSO-d₆): δ 11.32 (1H, s), 9.01–9.04 (2H, m), 8.83 (1H, s),8.45 (1H, d, J=8 Hz), 7.22–7.73 (5H, m), 5.95 (2H, s). HRMS (M+H)⁺found: 388.0674. Calcd for C₁₉H₁₃ClF₂N₃O₂: 388.0664.

Example 86-Amino-9-(3-chlorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide

Step (a): Ethyl 9-(3-chlorobenzyl)-6-nitro-9H-β-carboline-3-carboxylate.The title compound was prepared by alkylation of ethyl6-nitro-9H-β-carboline-3-carboxylate (prepared according to Settimj, et.al., J. Heterocycl. Chem., 25, 1391–1397 (1988)) with 3-chlorobenzylchloride in a manner similar to step (a) of example 7. LCMS (APCI,M+H⁺): 410.1/412.1=3/1.

Step (b): Ethyl 6-amino-9-(3-chlorobenzyl)-9H-β-carboline-3-carboxylate.A solution of ethyl9-(3-chlorobenzyl)-6-nitro-9H-β-carboline-3-carboxylate (2.7 g, 6.59mmol) and titanium(III) chloride (36 mL, 20% solution, 46.75 mmol) in amixture of acetic acid (54 mL), THF (180 mL), water (54 mL) and DMF (10mL) was stirred for 5 hours at ambient temperature. The reaction mixturewas quenched with water (200 mL), and extracted with ethyl acetate (100mL). The pH of the water layer was adjusted to 7 with saturated Na₂CO₃aqueous solution. Then, the resulting precipitate was filtered and driedin vacuo. The product was extracted from the precipitate with CHCl₃ in aSoxhlet extractor and purified by chromatography with ethyl acetate toprovide the title compound (0.70 g, yield 27.8%). ¹H NMR (DMSO-d₆): δ9.06 (s, 1H), 8.70 (s, 1H), 7.50 (d, 1H, J=8.0 Hz), 7.45 (s, 1H),7.27–7.31 (m, 3H), 7.09 (s, 1H), 7.01 (d, 1H, J=8.0 Hz), 5.75 (s, 2H),5.05 (s, 2H), 4.36 (q, 2H, J=7.0 Hz), 1.35 (t, 3H, J=7.0 Hz). LCMS(APCI, M+H⁺): 380.1/382.1=3/1.

Step (c):6-Amino-9-(3-chlorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide. To astirred solution of ethyl6-amino-9-(3-chlorobenzyl)-9H-β-carboline-3-carboxylate (139 mg, 0.37mmol) in methanol (15 mL) under a nitrogen atmosphere were added NaOH(2N, 0.7 mL) and H₂NOH (15 mL, 50 wt. % solution in H₂O, 0.23 mol). Thesuspension was stirred for 7 days at ambient temperature and thendiluted with H₂O (10 mL). The mixture was filtered, and the solid wasboiled in methanol (20 mL). After filtration, the title compound (36 mg,27%) was obtained. ¹H NMR (400 MHz, DMSO-d₆): δ 11.17 (1H, s), 8.90–8.95(2H, m), 8.55 (1H, s), 6.96–7.51 (7H, m), 5.72 (2H, s), 5.03 (2H, br,s). HRMS (M+H)⁺ found: 367.0961. Calcd for C₁₉H₁₆ClN₄O₂: 367.0962.

Example 99-(3-Chloro-2,6-difluorobenzyl)-N-methoxy-9H-β-carboline-3-carboxamide

The title compound is prepared by coupling of9-(3-chloro-2,6-difluorobenzyl)-9H-β-carboline-3-carboxylic acid andO-methyl hydroxylamine hydrochloride under conditions similar to thoseprovided in step (b) of example 7.

Example 10N-(Benzyloxy)-9-(3-chloro-2,6-difluorobenzyl)-9H-β-carboline-3-carboxamide

The title compound is prepared by coupling of9-(3-chloro-2,6-difluorobenzyl)-9H-β-carboline-3-carboxylic acid andO-benzyl hydroxyamine under conditions similar to those provided in step(b) of example 7.

Example 119-(3-Chloro-2,6-difluorobenzyl)-N-hydroxy-N-methyl-9H-□-carboline-3-carboxamide

The title compound is prepared by coupling of9-(3-chloro-2,6-difluorobenzyl)-9H-β-carboline-3-carboxylic acid andN-methyl hydroxylamine hydrochloride under conditions similar to thoseprovided in step (b) of example 7.

Example 12N-Benzyl-9-(3-chloro-2,6-difluorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide

The title compound is prepared by coupling of9-(3-chloro-2,6-difluorobenzyl)-9H-β-carboline-3-carboxylic acid andN-benzyl hydroxylamine hydrochloride under conditions similar to thoseprovided in step (b) of example 7.

Example 139-(4-Fluorobenzyl)-N-hydroxy-N-methyl-9H-β-carboline-3-carboxamide

The title compound can be prepared from ethyl9-(4-fluorobenzyl)-9H-β-carboline-3-carboxylate under the similarconditions as those in steps (a) and (b) of example 7. ¹H NMR (400 MHz,CD₃OD): δ 8.60–8.95 (2H, m), 8.32 (1H, d, J=8 Hz), 7.60–7.75 (2H, m),6.95–7.45 (5H, m), 5.76 (2H, s), 3.48 (3H, s). HRMS (M+H)⁺ found:350.1297. Calcd for C₂₀H₁₇FN₃O₂: 350.1305.

Example 14 Integrase Strand-Transfer Scintillation Proximity Assay

Oligonucleotides: Oligonucleotide#1—5′-(biotin)CCCCTTTTAGTCAGTGTGGAAAATCTCTAGCA-3′ (SEQ ID NO: 1) andoligonucleotide #2—5′-ACTGCTAGAGATTTTCCACACTGACTAAAAG-3′ (SEQ ID NO: 2),were synthesized by TriLink BioTechnologies, Inc. (San Diego, Calif.).The annealed product represents preprocessed viral ds-DNA derived fromthe LTR U5 sequence of the viral genome. A ds-DNA control to test fornon-specific interactions was made using a 3′ di-deoxy derivative ofoligonucleotide #1 annealed to oligonucleotide #2. The CA overhang atthe 5′ end of the non-biotinylated strand of the ds-DNA was createdartificially by using a complimentary DNA oligonucleotide shortened by 2base pairs. This configuration eliminates the requisite 3′ processingstep of the integrase enzyme prior to the strand-transfer mechanism.

Host ds-DNA was prepared as an unlabeled and [³H]-thymidine labeledproduct from annealed oligonucleotide #3—5-AAAAAATGACCAAGGGCTAATTCACT-3′(SEQ ID NO: 3), and oligonucleotide #4—5′-AAAAAAAGTGAATTAGCCCTTGGTCA-3′(SEQ ID NO: 4), both synthesized by TriLink BioTechnologies, Inc. (SanDiego, Calif.). The annealed product had overhanging 3′ ends ofpoly(dA). Host DNA was custom radiolabeled by PerkinElmer Life SciencesInc. (Boston, Mass.) using an enzymatic method with a 12/1 ratio of[methyl-3H]dTTP/cold ds-DNA to yield 5′-blunt end ds-DNA with a specificactivity of >900 Ci/mmol. The radiolabeled product was purified using aNENSORB cartridge and stored in stabilized aqueous solution(PerkinElmer). The final radiolabeled product had six [³H]-thymidinenucleotides at both 5′ ends of the host ds-DNA.

Reagents: Streptavidin-coated polyvinyltoluene (PVT) SPA beads werepurchased from Amersham Pharmacia (Piscataway, N.J.). Cesium chloridewas purchased from Shelton Scientific, Inc. (Shelton, Conn.). Whitepolystyrene, flat bottom, non-binding surface 384-well plates werepurchased from Corning. All other buffer components were purchased fromSigma (St. Louis, Mo.) unless otherwise indicated.

Enzyme Construction: Full-length HIV-1 integrase (SF1) sequence (aminoacids 1–288) (SEQ ID NO: 5) was constructed in a pET 15b vector(Novagen, Madison, Wis.) with mutations outlined by Chen et al. (Chen,C-H. J. et al., PNAS 97: 8233–8238 (2000)) that facilitate solubility ofthe enzyme and decrease oxidation. The vector contained a T7 promoter, a6-histidine tag at the amino terminus, and a thrombin cleavage site.Mutations C56S, W131D, F139D, F185K, and C280S were introduced using aQuickChange kit (Stratagene, San Diego, Calif.). The construct wasconfirmed through DNA sequencing.

Enzyme Purification: The penta-mutant was expressed in E. coli BL21(DE3) cells and induced with 1 mM isopropyl-1 thio-β-D-galactopyranoside(IPTG) when cells reached an optical density between 0.8–1.0 at 600 nm.Cells were lysed in 20 mM HEPES (pH 7.5), 1.5 M NaCl, 5 mM imidazole,and 2 mM 2-mercaptoethanol. The enzyme was purified following standardmethods for histidine tagged proteins (Jenkins, T. M. et al., Journal ofBiological Chemistry 271: 7712–7718 (1996)). Specifically, cell lysatewas passed over a Ni-Nta column (Qiagen, Chatsworth, Calif.) with the6-His tagged integrase protein eluted by adding 250 mM imidazole. A G-25Sepharose® column (Amersham Pharmacia, Piscataway, N.J.) was used toexchange the buffer prior to thrombin cleavage of the integrase proteinand subsequent removal of thrombin using a benzamidine-Sepharose® 6Bcolumn. The cleaved 6-His tag was separated from the integrase using asecond Ni-Nta column. The integrase was further purified with aheparin-Sepharose® column and a gradient of NaCl (0.4 to 1 M) in 20 mMHEPES (pH 7.5), 400 mM NaCl, and 1 mM DTT buffer. The purified proteinwas dialyzed against 20 mM HEPES (pH 7.5), 800 mM NaCl, and 1 mM DTT andconcentrated by stirred cell ultrafiltration (Millipore, Bedford, Mass.)or Ultra-free spin concentrators (Millipore, Bedford, Mass.) whenrequired. Viral DNA Bead Preparation: Streptavidin-coated SPA beads weresuspended to 20 mg/ml in 25 mM 3-morpholinopropanesulfonic acid (MOPS)(pH 7.2) and 0.1% NaN₃. Biotinylated viral DNA was bound to the hydratedSPA beads in a batch process by combining 25 pmoles of ds-DNA to 1 mg ofsuspended SPA beads (10 μl of 50 μM viral DNA to 1 ml of 20 mg/ml SPAbeads). The mixture was incubated at 22° C. for 20 min. with occasionalmixing followed by centrifugation at about 2500 rpm for about 10 min.However, the centrifugation speed and time may vary depending upon theparticular centrifuge and conditions. The supernatant was removed andthe beads suspended to 20 mg/ml in 25 mM MOPS (pH 7.2) and 0.1% NaN₃.The viral DNA beads were stable for more than 2 weeks when stored at 4°C. Di-deoxy viral DNA was prepared in an identical manner to yieldcontrol di-deoxy viral DNA beads.

Preparation of Integrase-DNA Complex: Assay buffer was made as a 10xstock of 250 mM MOPS (pH 7.2), 250 mM NaCl, 50 mM3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.5%(octylphenoxy)polyethoxyethanol (NP40) (IGEPAL-CA) and 0.05% NaN₃. ViralDNA beads were diluted to 2.67 mg/ml in 1× assay buffer plus 3 mM MgCl₂,1% DMSO, and 10 mM fresh DTT. Integrase (IN) was pre-complexed to viralDNA beads in a batch process (IN/viral DNA/bead complex) by combiningdiluted viral DNA beads with integrase at a concentration of 385 nMfollowed by a minimum incubation time of 15 min. at 22° C. The samplewas kept at 22° C. until transferred to the assay wells. Long-termstorage at 4° C. was possible, but not routinely applied.

Preparation of Host DNA: Host DNA was prepared to 200 nM as a mixture ofunlabeled and [³H]T-labeled host DNA diluted in 1x assay buffer plus 8.5mM MgCl₂ and 15 mM DTT. Typical concentrations were about 10 nM to about12 nM [³H]T-labeled host DNA and about 188 nM to about 190 nM unlabeledhost DNA. The ratio was adjusted relative to enzyme activity andspecific activity of the [³H]T-labeled host DNA to generate a SPA assaysignal of 2000–3000 CPM in the absence of modulators such as inhibitors.

Strand-transfer Scintillation Proximity Assay: The strand-transferreaction was carried out in 384-well microtiter plates, though anidentical protocol can be used for a 96-well plate format with a finalenzymatic reaction volume of 50 μl. Five microliters of compounds ortest reagents diluted in 10% DMSO were added to the assay wells followedby the addition of 32.5 μl of the IN/viral-DNA/bead complex. Thestrand-transfer reaction was initiated by adding 12.5 μl of host DNAwith vigorous vortexing of the plates and transferring them to ahumidified 37° C. incubator. An incubation time of 50 min. was shown tobe within the linear range of the enzymatic reaction in a 384-wellplate. Reaction kinetics are faster in a 96-well format. An incubationtime of 20 or 50 minutes was used as the time point to evaluate compoundinhibitors for assays run in the 96- or 384-well plate format,respectively. The final concentrations of integrase and host DNA in theassay wells were 246 nM and 50 nM, respectively.

The integrase strand-transfer reaction was terminated by adding 35 μl ofstop buffer (150 mM EDTA, 90 mM NaOH, and 6 M CsCl) to the wells.Components of the stop buffer function to terminate enzymatic activity(EDTA), dissociate integrase/DNA complexes in addition to separatingnon-integrated DNA strands (NaOH), and float the SPA beads to thesurface of the wells to be in closer range to the PMT detectors of theTopCount® plate-based scintillation counter (PerkinElmer Life SciencesInc. (Boston, Mass.)). After the addition of stop buffer, the plateswere vigorously vortexed, sealed with transparent tape, and allowed toincubate a minimum of 60 min. at 22° C. The assay signal was measuredusing a TopCount® plate-based scintillation counter with settingsoptimal for [3H]-PVT SPA beads. The TopCount® program incorporated aquench standardization curve to normalize data for color absorption ofthe compounds (color quench correction program (QstINT file). Datavalues for quench-corrected counts per minute (QCPM) were used toquantify integrase activity. Counting time was 30 sec./well for platesprocessed in HTS mode, and up to 2 min./well for plates containingpurified compound.

The di-deoxy viral DNA beads were used to optimize the integrasestrand-transfer reaction. The di-deoxy termination of the viral ds-DNAsequence prevented productive integration of viral DNA into the host DNAby integrase. Thus, the assay signal in the presence of di-deoxy viralDNA was a measure of non-specific interactions. Assay parameters wereoptimized to where reactions with di-deoxy viral DNA beads gave an assaysignal closely matched to the true background of the assay. The truebackground of the assay was defined as a reaction with all assaycomponents (viral DNA and [H³]-host DNA) in the absence of integrase.

Determination of Compound Activity: Compounds were evaluated forintegrase inhibitory activity using two different methods. Ahigh-throughput screening method was employed to test combinatorialcompound libraries or synthetic compounds that were solvated andtransferred to microtiter plates. The percent inhibition of the compoundwas calculated using the equation (1−((QCPM sample−QCPM min)/(QCPMmax−QCPM min)))*100. The min value is the assay signal in the presenceof a known inhibitor at a concentration 100-fold higher than the IC₅₀for that compound. The min signal approximates the true background forthe assay. The max value is the assay signal obtained for theintegrase-mediated activity in the absence of compound.

The IC₅₀ values of synthetic and purified combinatorial compounds werealso determined. Compounds were prepared in 100% DMSO at 100-fold higherconcentrations than desired for testing in assays, followed by dilutionof the compounds in 100% DMSO to generate an 8-point titration curvewith ½-log dilution intervals. The compound sample was further diluted10-fold with water and transferred to the assay wells. The percentageinhibition for an inhibitory compound was determined as above withvalues applied to a nonlinear regression, sigmoidal dose responseequation (variable slope) using GraphPad Prism curve fitting software(GraphPad Software, Inc., San Diego, Calif.).

Compound IC₅₀ (μM)* 1 0.234 2 0.494 3 0.4 4 0.281 5 9% inhibition at 50μM 6 0.463 7 1.39 8 0.713 13 0.699 *The error associated with each ofthese measurements is not shown

Example 14 HIV-1 Cell Protection Assay

The antiviral activities of potential modulator compounds (testcompounds) were determined in HIV-1 cell protection assays using the RFstrain of HIV-1, CEM-SS cells, and the XTT dye reduction method(Weislow, O. S. et al., J. Natl. Cancer Inst. 81: 577–586 (1989)).Subject cells were infected with HIV-1 RF virus at an moi of 0.025 to0.819 or mock infected with medium only and added at 2×10⁴ cells perwell into 96 well plates containing half-log dilutions of testcompounds. Six days later, 50 μl of XTT (1 mg/ml XTT tetrazolium and0.02 nM phenazine methosulfate) were added to the wells and the plateswere reincubated for four hours. Viability, as determined by the amountof XTT formazan produced, was quantified spectrophotometrically byabsorbance at 450 nm.

Data from CPE assays were expressed as the percent of formazan producedin compound-treated cells compared to formazan produced in wells ofuninfected, compound-free cells. The fifty percent effectiveconcentration (EC₅₀) was calculated as the concentration of compoundthat affected an increase in the percentage of formazan production ininfected, compound-treated cells to 50% of that produced by uninfected,compound-free cells. The 50% cytotoxicity concentration (CC₅₀) wascalculated as the concentration of compound that decreased thepercentage of formazan produced in uninfected, compound-treated cells to50% of that produced in uninfected, compound-free cells. The therapeuticindex was calculated by dividing the cytotoxicity (CC₅₀) by theantiviral activity (EC₅₀).

It is to be understood that the foregoing description is exemplary andexplanatory in nature, and is intended to illustrate the invention andits preferred embodiments. Thus, the scope of the invention should beunderstood to be defined not by the foregoing description, but by thefollowing claims and their equivalents.

1. A compound of formula (I),

wherein: R₁, R₂, R₃, R₄, R₅, and R₆ are independently selected fromhydrogen, halogen, C₁–C₆ alkyl, alkoxy C₁–C₆ alkyl, C₂–C₆ alkenyl, C₂–C₆alkynyl, —OR_(c), —NO₂, and —N(R_(c))₂; each R_(c) is independentlyselected from hydrogen, C₁–C₆ alkyl, C₂–C₆ alkenyl, and C₂–C₆ alkynyl;R₇ is C₁–C₆ alkyl, C₂–C₆ alkenyl, or C₂–C₆ alkynyl, all of which areoptionally substituted by one or more substituents independentlyselected from halogen, C₁–C₆ alkyl, C₂–C₆ alkenyl, C₂–C₆ alkynyl, aryl,cycloalkyl, heterocycloalkyl, and heteroaryl, wherein said aryl,cycloalkyl, and heterocycloalkyl are optionally substituted with one ormore substituents independently selected from halogen, C₁–C₆ alkyl,C₂–C₆ alkenyl, and C₂–C₆ alkynyl; R₈ and R₉ are independently selectedfrom hydrogen, C₁–C₆ alkyl, C₂–C₆ alkenyl, and C₂–C₆ alkynyl, whereinsaid alkyl, alkenyl, and alkynyl are optionally substituted with one ormore substituents independently selected from halogen, aryl, cycloalkyl,heterocycloalkyl, and heteroaryl group, wherein said aryl, cycloalkyl,and heterocycloalkyl are optionally substituted with one or moresubstituents independently selected from halogen, C₁–C₆ alkyl, C₂–C₆alkenyl, and C₂–C₆ alkynyl; and pharmaceutically acceptable salts andsolvates thereof.
 2. A compound according to claim 1, wherein: R₁, R₂,R₃, R₄, R₅, and R₆ are independently selected from hydrogen, —N(R_(c))₂,and —NO₂.
 3. A compound according to claim 1, wherein R₇ is C₁–C₆ alkyl,optionally substituted with aryl, cycloalkyl, heterocycloalkyl, andheteroaryl, wherein said aryl, cycloalkyl, heterocycloalkyl, andheteroaryl are optionally substituted with at least one substituentselected from halogen, C₁–C₆ alkyl, C₂–C₆ alkenyl, and C₂–C₆ alkynyl. 4.A compound according to claim 1, wherein R₈ and R₉ are independentlyselected from hydrogen and C₁–C₆ alkyl, wherein said alkyl group isoptionally substituted with aryl, and wherein said aryl is optionallysubstituted with at least one substituent selected from halogen andC₁–C₆ alkyl.
 5. A compound according to claim 1, wherein: R₁, R₂, R₃,R₄, R₅, R₆ are independently selected from hydrogen, —NH₂; and —NO₂; R₇is 4-fluorobenzyl, (5-chlorothien-2-yl)methyl, 3-chloro-2-fluorobenzyl,benzyl, 4-methylbenzyl, 2,4-difluorobenzyl, 3-chloro-2,6-difluorobenzyl,or 3-chlorobenzyl; and R₈ and R₉ are independently selected fromhydrogen, methyl, and benzyl.
 6. A compound according to claim 1,wherein: R₁, R₂, R₃, R₄, R₅ and R₆ are hydrogen; R₇ is —CH₂phenyl,wherein said phenyl is substituted with at least one substitutent chosenfrom fluorine and chlorine; R₈ is hydrogen or —CH₃; and R₉ is hydrogenor —CH₃.
 7. A compound according to claim 1, wherein: R₁, R₂, R₃, R₅ andR₆ are hydrogen; R₄ is —NO₂ or —NH₂; R₇ is —CH₂phenyl, wherein saidphenyl is substituted with at least one substitutent chosen fromfluorine and chlorine; R₈ is hydrogen or —CH₃; and R₉ is hydrogen or—CH₃.
 8. A compound according to claim 1, wherein: R₁, R₂, R₃, R₄, R₅and R₆ are hydrogen; R₇ is —CH₂phenyl, wherein said phenyl issubstituted with at least one substitutent chosen from fluorine andchlorine; and R₈ and R₉ are hydrogen.
 9. A compound according to claim1, wherein: R₁, R₂, R₃, R₄, R₅ and R₆ are hydrogen; R₇ is —CH₂phenyl,wherein said phenyl is substituted with at least one substitutent chosenfrom fluorine and chlorine; and R₈ and R₉ are —CH₃.
 10. A compoundaccording to claim 1, wherein: R₁, R₂, R₃, R₄, R₅ and R₆ are hydrogen;R₇ is —CH₂phenyl, wherein said phenyl is substituted with at least onesubstitutent chosen from fluorine and chlorine; R₈ is hydrogen; and R₉is —CH₃.
 11. A compound according to claim 1, wherein: R₁, R₂, R₃, R₄,R₅ and R₆ are hydrogen; R₇ is —CH₂phenyl, wherein said phenyl issubstituted with at least one substitutent chosen from fluorine andchlorine; R₈ is —CH₃; and R₉ is hydrogen.
 12. A compound according toclaim 1, selected from9-(4-fluorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide;9-[(5-chlorothien-2-yl)methyl]-N-hydroxy-9H-β-carboline-3-carboxamide;9-(3-chloro-2-fluorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide;9-Benzyl-N-hydroxy-9H-β-carboline-3-carboxamide;9-(4-methylbenzyl)-N-Hydroxy-9H-β-carboline-3-carboxamide;9-(2,4-difluorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide;9-(3-chloro-2,6-difluorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide;6-amino-9-(3-chlorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide;9-(3-chloro-2,6-difluorobenzyl)-N-methoxy-9H-β-carboline-3-carboxamide;N-(benzyloxy)-9-(3-chloro-2,6-difluorobenzyl)-9H-β-carboline-3-carboxamide;9-(3-chloro-2,6-difluorobenzyl)-N-hydroxy-N-methyl-9H-β-carboline-3-carboxamide;N-benzyl-9-(3-chloro-2,6-difluorobenzyl)-N-hydroxy-9H-β-carboline-3-carboxamide;9-(4-fluorobenzyl)-N-hydroxy-N-methyl-9Hβ-carboline-3-carboxamide; andpharmaceutically acceptable salts and solvates thereof.
 13. Apharmaceutical composition, comprising a therapeutically effectiveamount of at least one compound according to claim 1 and apharmaceutically acceptable carrier, diluent, or vehicle.
 14. Apharmaceutical composition, comprising a therapeutically effectiveamount of at least one compound according to claim 12 and apharmaceutically acceptable carrier, diluent, or vehicle.